WO2021051808A1 - 一种力刺激加载装置及其工作方法 - Google Patents

一种力刺激加载装置及其工作方法 Download PDF

Info

Publication number
WO2021051808A1
WO2021051808A1 PCT/CN2020/086396 CN2020086396W WO2021051808A1 WO 2021051808 A1 WO2021051808 A1 WO 2021051808A1 CN 2020086396 W CN2020086396 W CN 2020086396W WO 2021051808 A1 WO2021051808 A1 WO 2021051808A1
Authority
WO
WIPO (PCT)
Prior art keywords
torsion
force
gel
loading device
sun gear
Prior art date
Application number
PCT/CN2020/086396
Other languages
English (en)
French (fr)
Inventor
施媛萍
郑璐
罗光华
朱晓璐
王郑
尹曦
印强
Original Assignee
常州市第一人民医院
河海大学常州校区
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 常州市第一人民医院, 河海大学常州校区 filed Critical 常州市第一人民医院
Priority to US17/607,041 priority Critical patent/US20220204937A1/en
Publication of WO2021051808A1 publication Critical patent/WO2021051808A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0657—Cardiomyocytes; Heart cells
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B90/00—Instruments, implements or accessories specially adapted for surgery or diagnosis and not covered by any of the groups A61B1/00 - A61B50/00, e.g. for luxation treatment or for protecting wound edges
    • A61B90/06—Measuring instruments not otherwise provided for
    • 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
    • C12M23/04—Flat or tray type, drawers
    • 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
    • C12M25/02—Membranes; Filters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B90/00—Instruments, implements or accessories specially adapted for surgery or diagnosis and not covered by any of the groups A61B1/00 - A61B50/00, e.g. for luxation treatment or for protecting wound edges
    • A61B90/06—Measuring instruments not otherwise provided for
    • A61B2090/064—Measuring instruments not otherwise provided for for measuring force, pressure or mechanical tension
    • A61B2090/066—Measuring instruments not otherwise provided for for measuring force, pressure or mechanical tension for measuring torque

Definitions

  • the invention belongs to the medical-industrial cross-field, particularly the biomechanics and mechanical biology fields, and specifically relates to a force stimulation loading device and a working method thereof.
  • Cardiovascular disease is currently the leading cause of human death worldwide.
  • the development of myocardial tissue engineering provides the most potential solution for the treatment of cardiovascular disease.
  • cardiovascular disease During the occurrence and development of cardiovascular disease, it is closely related to the changes in the cellular power-the electrical microenvironment.
  • the mechanical microenvironment of in vivo cardiomyocytes will have various effects on the growth and signal transduction of cardiomyocytes. Changes in the mechanical microenvironment caused by diseases can also lead to abnormal physiological states of cardiomyocytes. Therefore, studying the influence of mechanical microenvironment on cells is also of great significance for exploring basic theories and the diagnosis and treatment of diseases.
  • the current research on the regulation of cell mechanics microenvironment is mainly to simulate the mechanical microenvironment of cells under normal physiological or pathological conditions by controlling the hardness or stiffness of two-dimensional or three-dimensional substrate materials; or to perform bionics on scaffold materials that wrap cells
  • the mechanical stretching stimulus can regulate the stress state of the cells at the micro-scale and promote the function of the cardiomyocytes.
  • the usual electrical stimulation loading is mainly achieved by designing various forms of electrodes to stimulate the cells in a pulsed manner. Studies have shown that cardiomyocytes seeded on conductive composite scaffolds have a significantly improved response to electrical stimulation, and can better conduct the applied electrical signals to promote the synchronized beating function of cardiomyocytes.
  • the force-electrical microenvironment is beneficial to improve the preparation process and functional simulation of engineered myocardial tissue.
  • the force signal stimulation or the force-electric coupling signal stimulation device Design and method optimization is an important part of the realization of mature engineered myocardial tissue.
  • the current research work on myocardial tissue engineering not only focuses on the selection and optimization of scaffold materials and seed cells, but also the systematic technology of promoting the function of engineered myocardial tissue through the regulation of cell force-electrical microenvironment has gradually become a hot spot for development. .
  • the main focus is on the morphology of cardiomyocytes, the expression of functional proteins and genes, and the frequency and intensity of synchronized contraction.
  • the changes in the elastic modulus of myocardial tissue are closely related to the changes in the function of myocardial cells.
  • cardiomyocytes can sense the static and dynamic mechanical stimuli in the cell microenvironment through the force-sensitive ion channels on the cell membrane, activate the electrophysiological and intracellular biochemical responses on the cell membrane, and realize the regulation of the structure and function of the cardiomyocytes.
  • Stem cells are usually sensitive to force, and mechanical stimuli such as tensile and compressive stress, shear stress, and stretch strain can affect the proliferation, skeletal structure and multidirectional differentiation process of stem cells.
  • mechanical stimuli such as tensile and compressive stress, shear stress, and stretch strain can affect the proliferation, skeletal structure and multidirectional differentiation process of stem cells.
  • shear stress generated by fluid flow plays an important role in embryonic development and organ formation, such as the activation and maturation of newborn cardiomyocytes, and the formation of zebrafish embryonic heart.
  • the purpose of the present invention is to provide a force stimulus loading device and its working method.
  • the present invention provides a force stimulus loading device, including: a receiving body, a stretching mechanism, and a torsion mechanism; wherein the receiving body is suitable for containing a gel that wraps myocardial cells and adopts a non-rigid material;
  • the stretching mechanism is adapted to stretch or squeeze the containing body from opposite sides of the containing body to apply a stretching force or a squeezing force to the gel; and the torsion mechanism is adapted to twist the containing body to apply a force to the gel. Apply torsion shear stress.
  • the accommodating body includes: an upper cover plate and a lower cover plate, and the upper and lower cover plates are connected by a clip cover; the inner surface of the upper cover plate is provided with a plurality of first protrusions at intervals; and A plurality of second protrusions are provided on the inner surface of the lower cover plate at intervals.
  • the stretching mechanism includes: a screw mechanism respectively symmetrically arranged on opposite sides of the containing body; the screw mechanism includes: a screw motor, a transmission shaft, a screw, and a nut; wherein the screw passes through The nut is provided, and one end is connected to the screw motor through the transmission shaft; the other end of the screw is connected to the clamping cover; each screw motor is adapted to drive the corresponding screw to move away or toward The direction of the clip is moved to stretch or squeeze the gel from opposite sides of the gel.
  • each nut is respectively located on a bracket.
  • the torsion mechanism is located on the upper cover through an upper splint, and includes: a torsion motor and a torsion assembly; wherein the torsion assembly includes: a housing, a sun gear, and several planetary gears meshing with the sun gear , And the outer rim meshed with the planet gears; the output shaft of the torsion motor is connected to the sun gear; the outer rim is fixed on the upper clamping plate; the gear shaft of the sun gear and the planets The gear shaft of the gear is fixed on the housing; and the housing is fixedly connected to a frame through a connecting rod; the torsion motor is adapted to drive the sun gear to drive the planet wheels to rotate, so as to drive the outer rim to rotate, thereby The upper splint drives the upper cover plate to rotate, thereby applying torsion shear stress to the gel.
  • the torsion assembly includes: a housing, a sun gear, and several planetary gears meshing with the sun gear , And the outer rim meshed with the planet gear
  • the torsion mechanism is located on the upper cover through an upper splint, and includes: a torsion motor and a torsion assembly; wherein the torsion assembly includes: a housing, a sun gear, and several planetary gears meshing with the sun gear , And the outer rim meshing with the planetary gears; the output shaft of the torsion motor is connected to the sun gear; the planet gears are fixed on the upper clamping plate; the gear shaft of the sun gear and the outer rim are fixed On the shell, and the shell is fixedly connected with a frame through a connecting rod; the torsion motor is suitable for driving the sun gear to drive the planet wheels to rotate, so as to drive the upper cover plate to rotate through the upper splint, thereby applying the gel Torsion shear stress.
  • the torsion assembly includes: a housing, a sun gear, and several planetary gears meshing with the sun gear , And the outer rim meshing with the planetary gears; the output shaft of the torsion motor is connected to the
  • the torsion motor is located on a support assembly; the support assembly includes: a cross bar and support rods respectively located at two ends of the cross bar.
  • a lower clamping plate is provided under the lower cover plate.
  • the present invention also provides a working method of a force stimulus loading device, which includes: stretching or squeezing the containing body from opposite sides of the containing body through a stretching mechanism to apply tension to the gel located in the containing body. Stretching force or squeezing force; and twisting the containing body through a torsion mechanism to apply torsion shear stress to the gel located in the containing body.
  • the beneficial effect of the present invention is that the force stimulus loading device of the present invention can simultaneously apply a stretching force, a squeezing force, and a torsion shear stress to the gel that wraps the cardiomyocytes through the stretching mechanism and the torsion mechanism, that is, it can simultaneously apply the tensile force to the cardiomyocytes.
  • Tensile force or squeezing force, torsion shear stress; this application also cooperates with the first protrusion on the upper cover plate and the second protrusion on the lower cover plate to facilitate the gel to interact with the upper and lower cover plates respectively It can greatly reduce the sliding deviation of the gel and ensure that the force can be evenly applied to the gel, that is, evenly applied to the cardiomyocytes.
  • Figure 1 is a schematic structural diagram of a force stimulus loading device according to an embodiment of the present invention (part of the support assembly is omitted);
  • Figure 2 is a schematic structural view of the force stimulus loading device according to the embodiment of the present invention from another angle (the stretching mechanism is omitted);
  • Fig. 3 is a schematic structural view of the torsion component (with the casing omitted) of the force stimulus loading device according to the embodiment of the present invention
  • Fig. 4 is a schematic diagram of a torsion state of the force stimulation loading device of the embodiment of the present invention.
  • Fig. 5 is a top view of the structure in which the housing of the torsion assembly according to the embodiment of the present invention is fixedly connected to the frame connecting rod through the connecting rod.
  • this embodiment 1 provides a force stimulus loading device, including: a receiving body, a stretching mechanism, and a torsion mechanism; wherein the receiving body is suitable for containing the gel 2 that wraps the cardiomyocyte 21 , And use a non-rigid material; the stretching mechanism is suitable for stretching or squeezing the containing body from opposite sides of the containing body to apply a stretching force or a pressing force to the gel 2; and the torsion mechanism is suitable for The accommodating body is twisted to apply torsion shear stress to the gel 2.
  • the force stimulus loading device of this embodiment can simultaneously apply a stretching force, a squeezing force, and a torsion shear stress to the gel 2 wrapping the cardiomyocytes 21 through the stretching mechanism and the torsion mechanism.
  • the accommodating body includes: an upper cover plate 1 and a lower cover plate 3, and the upper and lower cover plates are connected by a card cover 13; the inner surface of the upper cover plate 1 is provided with a plurality of first protrusions at intervals The raised portion 11; and the inner surface of the lower cover plate 3 is provided with a number of second protrusions 31 at intervals.
  • the material of the upper cover plate 1 and the lower cover plate 3 is, for example, but not limited to, polydimethylsiloxane (pdms) or polytetrafluoroethylene;
  • the card cover 13 is also, for example, but not limited to, polydimethylsiloxane (PDMS) or polytetrafluoroethylene.
  • the first protrusion 11 is for example but not limited to rectangular teeth
  • the second protrusion 31 is also for example but not limited to rectangular teeth
  • the gel 2 is clamped in Between the first and second protrusions, the cooperation of the first and second protrusions facilitates the adhesion of the gel 2 to the upper and lower cover plates respectively, and can greatly reduce the sliding deviation of the gel to ensure The force can be applied evenly to the gel.
  • the stretching mechanism includes: screw mechanisms respectively symmetrically arranged on opposite sides of the containing body; the screw mechanism includes: a screw motor (40; 50), a transmission shaft (41; 51), a wire Rod (42; 52) and a nut; wherein the screw rod (42; 52) passes through the nut, and one end is connected to the screw motor (40; 50) through the transmission shaft (41; 51); The other end of the screw rod (42; 52) is connected to the card cover 13; each screw motor (40; 50) is adapted to drive the corresponding screw rods (42; 52) to move away from or toward the card cover 13 Move in the direction to stretch or squeeze the gel 2 from opposite sides of the gel 2.
  • the screw mechanism includes: a screw motor (40; 50), a transmission shaft (41; 51), a wire Rod (42; 52) and a nut; wherein the screw rod (42; 52) passes through the nut, and one end is connected to the screw motor (40; 50) through the transmission shaft (41; 51); The other end of
  • the screw mechanism adopts a micro screw mechanism and is controlled by a control module;
  • the screw motor (40; 50) adopts a micro servo motor to improve the precision of stretching or extrusion; each screw motor ( 40; 50) respectively drive the corresponding screw rods to move away from or toward the card cover 13, so as to stretch or squeeze the gel 2 from opposite sides of the gel 2 to further ensure the stretching or squeezing force Apply evenly on gel 2.
  • each nut is respectively located on a bracket (43; 53).
  • the torsion mechanism is located on the upper cover plate 1 through an upper clamping plate 70, and includes: a torsion motor 84 and a torsion assembly 80; wherein the torsion assembly 80 includes: a housing 801, a central gear 81, and the central gear 81 A number of planetary gears 82 that mesh with each other, and a peripheral rim 83 that meshes with each of the planetary gears 82; the output shaft of the torsion motor 84 is connected to the sun gear 81; the peripheral rim 83 is fixed on the upper splint 70; the gear shaft of the sun gear 81 and the gear shafts of the planet gears 82 are fixed on the housing 801; the torsion motor 84 is adapted to drive the sun gear 81 to drive the planet gears 82 to rotate to drive the The outer rim 83 rotates, so that the upper splint 70 drives the upper cover 1 to rotate, thereby applying torsion shear stress to the gel 2.
  • the torsion assembly 80 includes: a
  • the rotation of the outer rim 83 drives the upper cover 1 to rotate, and the torsion shear stress is applied to the gel 2.
  • the rotation diameter of this embodiment is larger. .
  • the housing 801 is also fixedly connected to the corresponding rack connecting rods on the rack through connecting rods 931, connecting rods 932, connecting rods 933, and connecting rods 934, that is, connecting rods 931 and rack connecting rods 921 are fixedly connected.
  • the connecting rod 932 is fixedly connected to the frame connecting rod 922
  • the connecting rod 933 is fixedly connected to the frame connecting rod 923
  • the connecting rod 934 is fixedly connected to the frame connecting rod 924.
  • the torsion mechanism is located on the upper cover plate 1 through an upper clamping plate 70, and includes: a torsion motor 84 and a torsion assembly 80; wherein the torsion assembly 80 includes: a housing 801, a central gear 81, and the central gear 81 A number of planetary gears 82 meshing, and a peripheral rim 83 meshing with each planetary gear 82; the output shaft of the torsion motor 84 is connected to the sun gear 81; each planetary gear 82 is fixed on the upper clamping plate 70 The gear shaft of the sun gear 81 and the outer rim 83 are fixed on the housing 801; the torsion motor 84 is adapted to drive the sun gear 81 to drive the planetary gears 82 to rotate to drive the outer rim 83 to rotate , So that the upper cover plate 1 is driven to rotate by the upper splint 70, and the torsion shear stress is applied to the gel 2 accordingly.
  • the torsion assembly 80 includes: a housing 801, a central gear
  • each planetary gear 82 by fixing each planetary gear 82 on the upper splint 70, the rotation of each planetary gear 82 drives the upper cover 1 to rotate, and torsion shear stress is applied to the gel 2.
  • the rotation diameter of this embodiment is small. .
  • the housing 801 is also fixedly connected to the corresponding rack connecting rods on the rack through connecting rods 931, connecting rods 932, connecting rods 933, and connecting rods 934, that is, connecting rods 931 and rack connecting rods 921 are fixedly connected.
  • the connecting rod 932 is fixedly connected to the frame connecting rod 922
  • the connecting rod 933 is fixedly connected to the frame connecting rod 923
  • the connecting rod 934 is fixedly connected to the frame connecting rod 924.
  • a suitable torsion mechanism is selected according to the size of the biological sample and the force required to be loaded.
  • the torsion mechanism is also controlled by the control module; the gel 2 is twisted around the center of rotation 12, and the torsion motor 84 uses a micro servo motor to improve the torsion accuracy.
  • the torsion motor 84 is located on a support assembly; the support assembly includes: a cross bar 92 and support rods (91; 93) respectively located at two ends of the cross bar 92.
  • a lower clamping plate 60 is provided under the lower cover plate 3.
  • this embodiment 2 provides a working method of a force stimulus loading device, which includes: stretching or squeezing the receiving body from opposite sides of the receiving body through a stretching mechanism to align the receiving body The gel is stretched or squeezed; and the container is twisted by a twisting mechanism to apply torsion shear stress to the gel in the container.
  • the specific structure and principle of the force stimulus loading device can refer to the description of Embodiment 1, which will not be repeated here.
  • the force stimulus loading device can simultaneously apply tensile force, squeezing force, and torsion shear stress to the gel that wraps the cardiomyocytes through the stretching mechanism and the torsion mechanism, that is, it can simultaneously apply the stretching force or the torsion shear stress to the cardiomyocytes.
  • Squeezing force and torsion shear stress this application also facilitates the adhesion of the gel with the upper and lower cover plates through the cooperation of the first raised portion on the upper cover plate and the second raised portion on the lower cover plate, And it can greatly reduce the sliding deviation of the gel, and ensure that the force can be evenly applied to the gel, that is, evenly applied to the cardiomyocytes.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical & Material Sciences (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Sustainable Development (AREA)
  • Cell Biology (AREA)
  • Mechanical Engineering (AREA)
  • Surgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Rheumatology (AREA)
  • Immunology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Pathology (AREA)
  • Clinical Laboratory Science (AREA)
  • Cardiology (AREA)
  • Medical Informatics (AREA)
  • Molecular Biology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Prostheses (AREA)
  • Percussion Or Vibration Massage (AREA)

Abstract

本发明提供一种力刺激加载装置及其工作方法，该力刺激加载装置包括：容纳体、拉伸机构和扭转机构；该容纳体适于容纳包裹心肌细胞的凝胶，且采用非刚性材料；该拉伸机构适于从容纳体的相对两侧拉伸或挤压容纳体，以对凝胶施加拉伸力或挤压力；该扭转机构适于扭转容纳体，以对凝胶施加扭切应力；本发明的力刺激加载装置通过拉伸机构和扭转机构能够同时对包裹心肌细胞的凝胶施加拉伸力或挤压力、扭切应力，即能同时对心肌细胞施加拉伸力或挤压力、扭切应力。

Description

一种力刺激加载装置及其工作方法 技术领域
本发明属于医工交叉领域，特别是生物力学和力学生物领域，具体涉及一种力刺激加载装置及其工作方法。
背景技术
心血管疾病是当前全球范围内导致人类死亡的首要原因,心肌组织工程的发展为心血管疾病的治疗提供了最有潜力的解决方案。在心血管疾病的发生发展过程中，其与细胞力–电微环境的变化密切相关。近十几年,随着先进生物材料和微纳生物制造技术的发展,越来越多的研究表明,细胞力–电微环境的调控对工程化心肌组织的成熟和功能化以及心肌组织再生修复至关重要。在体心肌细胞所处力学微环境会对心肌细胞的生长及信号传导等产生多方面影响，由于疾病引起的力学微环境的变化亦会导致心肌细胞产生异常的生理状态。因此研究力学微环境对细胞的影响作用对于探究基础理论以及疾病的诊断治疗亦都具有重要意义。
目前对细胞力学微环境调控方面的研究,主要是通过控制二维或者三维基底材料的硬度或刚度等来模拟正常生理或病理状态下细胞所处力学微环境；或对包裹细胞的支架材料进行仿生力学拉伸刺激来调控细胞在微尺度下的受力状态进而促进心肌细胞的功能。通常的电刺激加载主要通过设计各种形式的电极对细胞进行脉冲式的刺激实现的。有研究表明，接种于导电的复合材料支架上的心肌细胞对电刺激的响应有较为明显的提高,能够更好地传导施加的电信号,以促进心肌细胞的同步搏动功能。因此,体外培养的过程中通过加载仿生力–电刺激重构细胞，力–电微环境利于改善工程化心肌组织的制备流程和功能模拟,其中对 力信号刺激或力–电耦合信号刺激装置的设计和方法优化是实现成熟的工程化心肌组织的重要内容。
目前有关心肌组织工程的研究工作不仅着眼于支架材料和种子细胞的选择和优化,而且对通过细胞力–电微环境的调控来促进工程化心肌组织功能方面的系统性技术也逐渐成为开发的热点。目前，在体外借助力学和电学刺激改善心肌细胞功能方面,主要关注心肌细胞形态、功能性蛋白和基因的表达以及同步收缩频率和强度等。心肌组织弹性模量的变化与心肌细胞功能改变密切相关,例如研究者发现细胞外基质的硬度不仅影响心肌细胞内的主动收缩力,而且会影响收缩应变,并会影响心肌细胞的搏动频率。同时,心肌细胞的基因、蛋白表达以及细胞间通讯等也被证实会受到心肌组织力学微环境的影响。心肌细胞能够通过细胞膜上的力敏感离子通道感受细胞微环境中的静态和动态力学刺激,激活细胞膜上的电生理和细胞内相关联的生化响应,进而实现对心肌细胞的结构和功能的调控。此外，在诱导骨髓间充质干细胞分化成心肌细胞并构建心肌组织方面，力学因素也发挥重要作用。干细胞通常对力比较敏感，拉压应力、剪切应力、牵张应变等力学刺激可以影响干细胞的增殖、骨架结构形态和多向分化过程。其中由于液体流动产生的剪切应力在胚胎发育和器官形成过程中起重要的作用，如新生心肌细胞的活化和成熟，以及斑马鱼胚胎心脏的形成等。
在研究力-电耦合环境下，心肌细胞的生理特性响应时，通常需要特定的激励施加及细胞功能测试装置，而目前的装置多为单一型，且在凝胶内细胞培养方面存在力学激励施加不均匀及难以同时实现拉伸及扭切应力等问题。
发明内容
本发明的目的是提供一种力刺激加载装置及其工作方法。
为了解决上述技术问题，本发明提供了一种力刺激加载装置，包括：容纳 体、拉伸机构和扭转机构；其中所述容纳体适于容纳包裹心肌细胞的凝胶，且采用非刚性材料；所述拉伸机构适于从容纳体的相对两侧拉伸或挤压容纳体，以对凝胶施加拉伸力或挤压力；以及所述扭转机构适于扭转容纳体，以对凝胶施加扭切应力。
进一步，所述容纳体包括：上盖板和下盖板，且上、下盖板之间通过一卡罩相连；所述上盖板的内表面上间隔设置有若干第一凸起部；以及所述下盖板的内表面上间隔设置有若干第二凸起部。
进一步，所述拉伸机构包括：分别对称设置在所述容纳体相对两侧的丝杆机构；所述丝杆机构包括：丝杆电机、传动轴、丝杆和螺母；其中所述丝杆穿设所述螺母，且一端通过所述传动轴与所述丝杆电机相连；所述丝杆的另一端与所述卡罩相连；各丝杆电机适于分别驱动相应的丝杆以远离或朝向卡罩的方向移动，以从凝胶的相对两侧拉伸或挤压所述凝胶。
进一步，各螺母分别位于一支架上。
进一步，所述扭转机构通过一上夹板位于所述上盖板上，且包括：扭转电机和扭转组件；其中所述扭转组件包括：壳体，中心齿轮，与该中心齿轮相啮合的若干行星齿轮，以及与各行星齿轮相啮合的外围轮圈；所述扭转电机的输出轴与所述中心齿轮相连；所述外围轮圈固定在所述上夹板上；所述中心齿轮的齿轮轴和各行星齿轮的齿轮轴固定在所述壳体上；且壳体通过连接杆与一机架固连；所述扭转电机适于驱动中心齿轮带动各行星轮转动，以带动所述外围轮圈转动，从而通过上夹板带动上盖板转动，进而对凝胶施加扭切应力。
进一步，所述扭转机构通过一上夹板位于所述上盖板上，且包括：扭转电机和扭转组件；其中所述扭转组件包括：壳体，中心齿轮，与该中心齿轮相啮合的若干行星齿轮，以及与各行星齿轮相啮合的外围轮圈；所述扭转电机的输 出轴与所述中心齿轮相连；各行星齿轮固定在所述上夹板上；所述中心齿轮的齿轮轴和外围轮圈固定在所述壳体上，且壳体通过连接杆与一机架固连；所述扭转电机适于驱动中心齿轮带动各行星轮转动，以通过上夹板带动上盖板转动，从而对凝胶施加扭切应力。
进一步，所述扭转电机位于一支撑组件上；所述支撑组件包括：横杆和分别位于所述横杆两端的支杆。
进一步，所述下盖板的下方设有下夹板。
又一方面，本发明还提供了一种力刺激加载装置的工作方法，包括：通过拉伸机构从容纳体的相对两侧拉伸或挤压容纳体，以对位于容纳体内的凝胶施加拉伸力或挤压力；以及通过扭转机构扭转容纳体，以对位于容纳体内的凝胶施加扭切应力。
本发明的有益效果是，本发明的力刺激加载装置通过拉伸机构和扭转机构能够同时对包裹心肌细胞的凝胶施加拉伸力或挤压力、扭切应力，即能同时对心肌细胞施加拉伸力或挤压力、扭切应力；本申请还通过上盖板上的第一凸起部与下盖板上的第二凸起部的配合，便于凝胶分别与上、下盖板的粘合，且能大大减小凝胶滑动偏移，确保力能够均匀施加到凝胶上，即均匀施加到心肌细胞上。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1是本发明实施例的力刺激加载装置的结构示意图(省略部分支撑组件)；
图2是本发明实施例的力刺激加载装置另一角度的结构示意图(省略拉伸机构)；
图3是本发明实施例的力刺激加载装置的扭转组件(省略壳体)的结构示 意图；
图4是本发明实施例的力刺激加载装置的的扭转状态示意图。
图5是本发明实施例的扭转组件的壳体通过连接杆与机架连杆固连的结构俯视图。
其中：
上盖板1，第一凸起部11，旋转中心12，卡罩13，凝胶2，心肌细胞21，下盖板3，第二凸起部31，丝杆电机40、50，传动轴41、51，丝杆42、52，支架43、53，下夹板60，上夹板70，扭转组件80，扭转组件的壳体801，中心齿轮81，行星齿轮82，外围轮圈83，扭转电机84，支杆91，横杆92，支杆93，连接杆931、932、933、934，机架连杆921、922、923、924。
具体实施方式
现在结合附图对本发明的结构作进一步详细的说明。
实施例1
如图1至图5所示，本实施例1提供了一种力刺激加载装置，包括：容纳体、拉伸机构和扭转机构；其中所述容纳体适于容纳包裹心肌细胞21的凝胶2，且采用非刚性材料；所述拉伸机构适于从容纳体的相对两侧拉伸或挤压容纳体，以对凝胶2施加拉伸力或挤压力；以及所述扭转机构适于扭转容纳体，以对凝胶2施加扭切应力。
具体的，本实施例的力刺激加载装置通过拉伸机构和扭转机构能够同时对包裹心肌细胞21的凝胶2施加拉伸力或挤压力、扭切应力。
进一步，所述容纳体包括：上盖板1和下盖板3，且上、下盖板之间通过一卡罩13相连；所述上盖板1的内表面上间隔设置有若干第一凸起部11；以及所述下盖板3的内表面上间隔设置有若干第二凸起部31。
具体的，上盖板1和下盖板3的材料例如但不限于采用聚二甲基硅氧烷(pdms)或者聚四氟乙烯；所述卡罩13也例如但不限于采用聚二甲基硅氧烷(pdms)或者聚四氟乙烯；第一凸起部11例如但不限于为矩形齿；第二凸起部31也例如但不限于采用矩形齿；所述凝胶2被夹持在第一、第二凸起部之间，通过第一、第二凸起部的配合，便于凝胶2分别与上、下盖板的粘合，且能大大减小凝胶滑动偏移，确保力能够均匀施加到凝胶上。
进一步，所述拉伸机构包括：分别对称设置在所述容纳体相对两侧的丝杆机构；所述丝杆机构包括：丝杆电机(40；50)、传动轴(41；51)、丝杆(42；52)和螺母；其中所述丝杆(42；52)穿设所述螺母，且一端通过所述传动轴(41；51)与所述丝杆电机(40；50)相连；所述丝杆(42；52)的另一端与所述卡罩13相连；各丝杆电机(40；50)适于分别驱动相应的丝杆(42；52)以远离或朝向卡罩13的方向移动，以从凝胶2的相对两侧拉伸或挤压所述凝胶2。
具体的，所述丝杆机构采用微型丝杆机构，并由一控制模块控制；所述丝杆电机(40；50)采用微型伺服电机，以提高拉伸或挤压精度；各丝杆电机(40；50)分别驱动相应的丝杆以远离或朝向卡罩13的方向移动，实现从凝胶2的相对两侧拉伸或挤压所述凝胶2，进一步确保拉伸力或挤压力均匀施加在凝胶2上。
进一步，各螺母分别位于一支架(43；53)上。
作为本实施例的扭转机构的第一种实施方式：
所述扭转机构通过一上夹板70位于所述上盖板1上，且包括：扭转电机84和扭转组件80；其中所述扭转组件80包括：壳体801，中心齿轮81，与该中心齿轮81相啮合的若干行星齿轮82，以及与各行星齿轮82相啮合的外围轮圈83；所述扭转电机84的输出轴与所述中心齿轮81相连；所述外围轮圈83固定在所 述上夹板70上；所述中心齿轮81的齿轮轴和各行星齿轮82的齿轮轴固定在所述壳体801上；所述扭转电机84适于驱动中心齿轮81带动各行星轮82转动，以带动所述外围轮圈83转动，从而通过上夹板70带动上盖板1转动，进而对凝胶2施加扭切应力。
具体的，通过将外围轮圈83固定在所述上夹板70上，实现通过外围轮圈83的转动带动上盖板1转动，对凝胶2施加扭切应力，本实施方式的旋转直径较大。
具体的，所述壳体801还分别通过连接杆931、连接杆932、连接杆933和连接杆934与机架上的相应机架连杆固连，即连接杆931与机架连杆921固连、连接杆932与机架连杆922固连、连接杆933与机架连杆923固连以及连接杆934与机架连杆924固连。
作为本实施例的扭转机构的第二种实施方式：
所述扭转机构通过一上夹板70位于所述上盖板1上，且包括：扭转电机84和扭转组件80；其中所述扭转组件80包括：壳体801，中心齿轮81，与该中心齿轮81相啮合的若干行星齿轮82，以及与各行星齿轮82相啮合的外围轮圈83；所述扭转电机84的输出轴与所述中心齿轮81相连；各行星齿轮82固定在所述上夹板70上；所述中心齿轮81的齿轮轴和外围轮圈83固定在所述壳体801上；所述扭转电机84适于驱动中心齿轮81带动各行星轮82转动，以带动所述外围轮圈83转动，从而通过上夹板70带动上盖板1转动，进而对凝胶2施加扭切应力。
具体的，通过将各行星齿轮82固定在所述上夹板70上，实现通过各行星齿轮82的转动带动上盖板1转动，对凝胶2施加扭切应力，本实施方式的旋转直径较小。
具体的，所述壳体801还分别通过连接杆931、连接杆932、连接杆933和连接杆934与机架上的相应机架连杆固连，即连接杆931与机架连杆921固连、连接杆932与机架连杆922固连、连接杆933与机架连杆923固连以及连接杆934与机架连杆924固连。
在实际应用中，根据生物样品的尺寸和所需加载的力的大小选择合适的扭转机构。
具体的，所述扭转机构也由所述控制模块控制；凝胶2围绕旋转中心12扭转，所述扭转电机84采用微型伺服电机，以提高扭转精度。
进一步，所述扭转电机84位于一支撑组件上；所述支撑组件包括：横杆92和分别位于所述横杆92两端的支杆(91；93)。
进一步，所述下盖板3的下方设有下夹板60。
实施例2
在实施例1的基础上，本实施例2提供了一种力刺激加载装置的工作方法，包括：通过拉伸机构从容纳体的相对两侧拉伸或挤压容纳体，以对位于容纳体内的凝胶施加拉伸力或挤压力；以及通过扭转机构扭转容纳体，以对位于容纳体内的凝胶施加扭切应力。
具体的，所述力刺激加载装置的具体结构及原理可参考实施例1的描述，此处不再赘述。
综上所述，本力刺激加载装置通过拉伸机构和扭转机构能够同时对包裹心肌细胞的凝胶施加拉伸力或挤压力、扭切应力，即能同时对心肌细胞施加拉伸力或挤压力、扭切应力；本申请还通过上盖板上的第一凸起部与下盖板上的第二凸起部的配合，便于凝胶分别与上、下盖板的粘合，且能大大减小凝胶滑动偏移，确保力能够均匀施加到凝胶上，即均匀施加到心肌细胞上。
以上述依据本发明的理想实施例为启示，通过上述的说明内容，相关工作人员完全可以在不偏离本项发明技术思想的范围内，进行多样的变更以及修改。本项发明的技术性范围并不局限于说明书上的内容，必须要根据权利要求范围来确定其技术性范围。

Claims (9)

  1. 一种力刺激加载装置，其特征在于，包括：
    容纳体、拉伸机构和扭转机构；其中
    所述容纳体适于容纳包裹心肌细胞的凝胶，且采用非刚性材料；
    所述拉伸机构适于从容纳体的相对两侧拉伸或挤压容纳体，以对凝胶施加拉伸力或挤压力；以及
    所述扭转机构适于扭转容纳体，以对凝胶施加扭切应力。
  2. 根据权利要求1所述的力刺激加载装置，其特征在于，
    所述容纳体包括：上盖板和下盖板，且上、下盖板之间通过一卡罩相连；
    所述上盖板的内表面上间隔设置有若干第一凸起部；以及
    所述下盖板的内表面上间隔设置有若干第二凸起部。
  3. 根据权利要求2所述的力刺激加载装置，其特征在于，
    所述拉伸机构包括：分别对称设置在所述容纳体相对两侧的丝杆机构；
    所述丝杆机构包括：丝杆电机、传动轴、丝杆和螺母；其中
    所述丝杆穿设所述螺母，且一端通过所述传动轴与所述丝杆电机相连；
    所述丝杆的另一端与所述卡罩相连；
    各丝杆电机适于分别驱动相应的丝杆以远离或朝向卡罩的方向移动，以从凝胶的相对两侧拉伸或挤压所述凝胶。
  4. 根据权利要求3所述的力刺激加载装置，其特征在于，
    各螺母分别位于一支架上。
  5. 根据权利要求2所述的力刺激加载装置，其特征在于，
    所述扭转机构通过一上夹板位于所述上盖板上，且包括：扭转电机和扭转组件；其中
    所述扭转组件包括：壳体，中心齿轮，与该中心齿轮相啮合的若干行星齿 轮，以及与各行星齿轮相啮合的外围轮圈；
    所述扭转电机的输出轴与所述中心齿轮相连；
    所述外围轮圈固定在所述上夹板上；
    所述中心齿轮的齿轮轴和各行星齿轮的齿轮轴固定在所述壳体上，且壳体通过连接杆与一机架固连；
    所述扭转电机适于驱动中心齿轮带动各行星轮转动，以带动所述外围轮圈转动，从而通过上夹板带动上盖板转动，进而对凝胶施加扭切应力。
  6. 根据权利要求2所述的力刺激加载装置，其特征在于，
    所述扭转机构通过一上夹板位于所述上盖板上，且包括：扭转电机和扭转组件；其中
    所述扭转组件包括：壳体，中心齿轮，与该中心齿轮相啮合的若干行星齿轮，以及与各行星齿轮相啮合的外围轮圈；
    所述扭转电机的输出轴与所述中心齿轮相连；
    各行星齿轮固定在所述上夹板上；
    所述中心齿轮的齿轮轴和外围轮圈固定在所述壳体上，且壳体通过连接杆与一机架固连；
    所述扭转电机适于驱动中心齿轮带动各行星轮转动，以通过上夹板带动上盖板转动，从而对凝胶施加扭切应力。
  7. 根据权利要求5或6所述的力刺激加载装置，其特征在于，
    所述扭转电机位于一支撑组件上；
    所述支撑组件包括：横杆和分别位于所述横杆两端的支杆。
  8. 根据权利要求2所述的力刺激加载装置，其特征在于，
    所述下盖板的下方设有下夹板。
  9. 一种力刺激加载装置的工作方法，其特征在于，包括：
    通过拉伸机构从容纳体的相对两侧拉伸或挤压容纳体，以对位于容纳体内的凝胶施加拉伸力或挤压力；以及
    通过扭转机构扭转容纳体，以对位于容纳体内的凝胶施加扭切应力。
PCT/CN2020/086396 2019-09-16 2020-04-23 一种力刺激加载装置及其工作方法 WO2021051808A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/607,041 US20220204937A1 (en) 2019-09-16 2020-04-23 Force Stimulation Loading Device and Working Method Thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910869033.2A CN110484426A (zh) 2019-09-16 2019-09-16 一种力刺激加载装置及其工作方法
CN201910869033.2 2019-09-16

Publications (1)

Publication Number Publication Date
WO2021051808A1 true WO2021051808A1 (zh) 2021-03-25

Family

ID=68558046

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/086396 WO2021051808A1 (zh) 2019-09-16 2020-04-23 一种力刺激加载装置及其工作方法

Country Status (3)

Country Link
US (1) US20220204937A1 (zh)
CN (1) CN110484426A (zh)
WO (1) WO2021051808A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114752494A (zh) * 2022-03-24 2022-07-15 四川大学 细胞培养室、可调式细胞力学刺激培养装置及其制作方法

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110484426A (zh) * 2019-09-16 2019-11-22 常州市第一人民医院 一种力刺激加载装置及其工作方法
CN110964637B (zh) * 2019-12-30 2020-11-06 北京航空航天大学 一种体外动态细胞培养装置及其培养方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101265466A (zh) * 2008-04-30 2008-09-17 天津理工大学 复合载荷下用于有冠状曲面组织的培养方法及生物反应器
CN101892152A (zh) * 2010-08-03 2010-11-24 北京航空航天大学 一种牵张-电联合刺激细胞培养装置
CN103740590A (zh) * 2013-12-13 2014-04-23 西安交通大学 一种三维细胞力学梯度加载平台
CN106795509A (zh) * 2014-03-06 2017-05-31 加利福尼亚大学董事会 用于测量细胞机械应力的组合物和方法
CN110484426A (zh) * 2019-09-16 2019-11-22 常州市第一人民医院 一种力刺激加载装置及其工作方法
CN110551854A (zh) * 2019-09-16 2019-12-10 常州市第一人民医院 采用力刺激方式进行心肌细胞体外功能测试与调控的方法

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100799988B1 (ko) * 2006-11-30 2008-01-31 연세대학교 산학협력단 줄기세포 배양을 위하여 다양한 기계적 자극을 가하는 세포 배양 장치 및 그것을 이용한 줄기세포 배양방법
WO2012017343A2 (en) * 2010-08-05 2012-02-09 Koninklijke Philips Electronics N.V. Cardiomyocyte containing device, manufacturing method and measuring method
CN201883096U (zh) * 2010-11-09 2011-06-29 天津理工大学 一种双频压缩-径向扭转椎间盘组织工程生物反应器
CN102796664A (zh) * 2011-05-27 2012-11-28 长江大学 一种人体韧带组织工程生物反应器
CN102433258B (zh) * 2011-12-01 2014-03-12 北京航空航天大学 一种牵张-电联合刺激三维细胞培养装置
KR101506806B1 (ko) * 2013-09-23 2015-03-30 중앙대학교 산학협력단 세포 배양 장치
US20150093823A1 (en) * 2013-10-02 2015-04-02 President And Fellows Of Harvard College Environmentally Responsive Microstructured Hybrid Actuator Assemblies For Use in Mechanical Stimulation of Cells
KR101506811B1 (ko) * 2013-10-04 2015-03-30 중앙대학교 산학협력단 세포 배양 장치
CN107988067A (zh) * 2017-11-08 2018-05-04 西安外事学院 一种基于组织特定形状的三维细胞梯度力学加载实验平台
CN211079122U (zh) * 2019-09-16 2020-07-24 常州市第一人民医院 一种力刺激加载装置

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101265466A (zh) * 2008-04-30 2008-09-17 天津理工大学 复合载荷下用于有冠状曲面组织的培养方法及生物反应器
CN101892152A (zh) * 2010-08-03 2010-11-24 北京航空航天大学 一种牵张-电联合刺激细胞培养装置
CN103740590A (zh) * 2013-12-13 2014-04-23 西安交通大学 一种三维细胞力学梯度加载平台
CN106795509A (zh) * 2014-03-06 2017-05-31 加利福尼亚大学董事会 用于测量细胞机械应力的组合物和方法
CN110484426A (zh) * 2019-09-16 2019-11-22 常州市第一人民医院 一种力刺激加载装置及其工作方法
CN110551854A (zh) * 2019-09-16 2019-12-10 常州市第一人民医院 采用力刺激方式进行心肌细胞体外功能测试与调控的方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114752494A (zh) * 2022-03-24 2022-07-15 四川大学 细胞培养室、可调式细胞力学刺激培养装置及其制作方法

Also Published As

Publication number Publication date
CN110484426A (zh) 2019-11-22
US20220204937A1 (en) 2022-06-30

Similar Documents

Publication Publication Date Title
WO2021051808A1 (zh) 一种力刺激加载装置及其工作方法
CN110551854B (zh) 采用力刺激方式进行心肌细胞体外功能测试与调控的方法
Boudou et al. A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues
Castro et al. Physically active bioreactors for tissue engineering applications
Visone et al. Cardiac meets skeletal: what’s new in microfluidic models for muscle tissue engineering
JP4758314B2 (ja) 骨格筋の培養用足場、同培養装置及び骨格筋の形成方法
Feng et al. An electro-tensile bioreactor for 3-D culturing of cardiomyocytes
Zhang et al. Dynamic model for characterizing contractile behaviors and mechanical properties of a cardiomyocyte
CN101906379A (zh) 模拟体内环境下可视化细胞精密拉伸装置
Naskar et al. Reprogramming the stem cell behavior by shear stress and electric field stimulation: lab-on-a-chip based biomicrofluidics in regenerative medicine
CN104046564B (zh) 一种仿生理环境力学刺激式生物反应器系统
CN211079122U (zh) 一种力刺激加载装置
Zhao et al. In vitro cell stretching devices and their applications: From cardiomyogenic differentiation to tissue engineering
EP3896149A1 (en) Modular magnetically driven bioreactor for cellular cultures and biomedical applications
Melo-Fonseca et al. Mechanical stimulation devices for mechanobiology studies: a market, literature, and patents review
CN201737929U (zh) 模拟体内环境下可视化细胞精密拉伸装置
CN205188307U (zh) 一种模拟口腔咀嚼力学环境的细胞培养装置
EP1990402A1 (en) Bioreactor to apply mechanical forces as an anabolic stimulus
Somers et al. Protocol for the Use of a Novel Bioreactor System for Hydrated Mechanical Testing, Strained Sterile Culture, and Force of Contraction Measurement of Tissue Engineered Muscle Constructs
EP3470511A1 (en) Bioreactor for the recellularization and mechanical stimulation of a diaphragm
CN104007029A (zh) 组织工程支架动态力学实验装置及方法
CN204058481U (zh) 一种神经轴突牵拉生长装置
Béland et al. Development of an open hardware bioreactor for optimized cardiac cell culture integrating programmable mechanical and electrical stimulations
CN103966094B (zh) 一种作用于细胞拉伸应力的培养装置及方法
Santoni et al. Microsystems for electromechanical stimulations to engineered cardiac tissues

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20864452

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20864452

Country of ref document: EP

Kind code of ref document: A1