• 专利附录
  • 专利说明
  • 权利要求
  • 类似技术
  • 同族专利
  • 引用文献
  • 法律状态
  • 优先权
    • 专利摘要:
    The invention relates to a microbioreactor module comprising a cultivation container, a holding tube, an asymmetric plug, and a casing. The cultivation container is attached to one end of the support tube, which encloses an outlet and supply line, which allows sampling as well as supplying cells or bioactive molecules and nutrients into the cultivation container. The cultivation container has an outer shell made of a semipermeable membrane material, which encloses a biocompatible support material or a framework made of a biocompatible support material. The plug is located at the other end of the support tube, seals the microbioreactor module to the outside, and has an asymmetric thickness to open or close openings in the wall of the casing depending on the positioning of the asymmetric plug. The invention also relates to a multimicrobioreactor, consisting of several such microbioreactor modules, which are arranged in parallel in a common cultivation space, in particular a reactor vessel (12) filled with medium, in which homogenization can take place by means of a pump element (13) operated by compressed air.
    公开号:CH714398B1
    申请号:CH00497/19
    申请日:2017-10-06
    公开日:2021-12-15
    发明作者:Yoen Ok Roth Dr
    申请人:Yoen Ok Roth Dr；
    IPC主号:
    专利说明:

    [0001] The use of stem cells is becoming increasingly important in medical research, particularly in the field of regenerative medicine. The cultivation of stem cells is also used in pharmaceutical research and in the cosmetics industry in some areas, for example to carry out ADME/Tox studies, i.e. to test new potential active substances for their properties in relation to absorption, distribution, metabolism, excretion and toxicity.
    [0002] Embryonic and so-called induced pluripotent stem cells are characterized by their almost infinite potential for self-renewal, proliferation and differentiation, also in cell culture. However, tailor-made methods and devices for cultivation play a decisive role in reliably directing differentiation towards a specific tissue type and at the same time preventing unwanted malignant formation of tumors (cf. Lutolf, MP; Gilbert, PM; Blau, HM, Designing materials to direct stem-cell fate.Nature 2009, 462 (7272), 433-441).
    Especially for use in regenerative medicine, there is great interest in cellular material obtained in vitro, which has a high degree of consistent quality, so that there is a great need for standardized cell culture methods that allow long-term cultivation and long-term differentiation of stem cells ensure controlled conditions and ideally enable process automation or at least process parallelization.
    There is therefore an intensive search for solutions for the targeted cultivation of various cell types from pluripotent stem cells. The first, sometimes very complex options and devices were developed for tissue production. In particular, so-called organ chips are used, which enable the cultivation of e.g. lung, liver, heart, skin or bronchial tissue (cf. Lang, Q.; Ren, Y.; Wu, Y.; Guo, Y, Zhao, X, Tao, Y, Liu, J, Zhao, H, Lei, L, Jiang, H. A multifunctional resealable perfusion chip for cell culture and tissue engineering.RSC Advances 2016, 6 (32), 27183-27190).
    [0005] In vivo, stem cells are found in tissue-specific stem cell niches of the body. In such a niche, the stem cells are not only physically bound, but the special microenvironment of the niche determines the development of the stem cells through its regulatory network of biochemical processes and signals, induced by chemokines, cytokines, growth factors, transmembrane receptors, and the extracellular matrix. For the cultivation of stem cells in vitro, attempts are therefore being made to artificially imitate the microenvironment of the stem cell niches (cf. Lutolf, MP; Gilbert, PM; Blau, HM, Designing materials to direct stem-cell fate. Nature 2009, 462 (7272), 433-441).
    In the imitation of the natural environment of stem cell niches, in addition to the cellular microenvironment, other cell types, as well as gradients and concentration gradients in the medium, play a major role with regard to the effectiveness of the process.
    [0007] EP 2 181 188 B1 discloses a microbioreactor which is arranged as a microfluidic system and is suitable for the cultivation of advanced cell cultures, in particular 3D cell cultures and stem cell cultures. A special feature is the construction of a media circuit for the perfusion of the microbioreactor. The sample carrier on which the cell growth takes place is one or more stacked 3D CellChips. By arranging several mutually independent microbioreactors on a microtiter plate, the possibility of a multimicrobioreactor, in particular for high-throughput screening, is given.
    An artificial stem cell niche is known from US 2011/0136226 A1, which comprises a rotating culture chamber in which a framework with mesenchymal connective tissue stem cells is attached, on which umbilical cord blood stem cells are cultivated. In this case, the culture chamber is supplied via a fluid supply system, in which the nutrient supply and the gas and waste exchange takes place through a dialysis membrane and a second fluid system enables cells to be harvested from the suspension inside the culture chamber.
    An artificial microenvironment is known from US 2011/0207166 A1, which corresponds to a replica of a niche in the bone marrow microenvironment, which consists of a framework covered with mesenchymal stem cells and a culture medium that allows the stem cells to proliferate in the culture . The artificial niche is suitable for the cultivation of hematopoietic and leukemic cells. The framework here consists of a net-like, stretchable matrix made of an elastomeric material, e.g. polycarbonate or polyurethane.
    [0010] However, the aforementioned known bioreactors all have the disadvantage that their structure is very complex and thus simple handling, production and in particular use as a one-way system is limited.
    [0011] DE 10 2014 001 615.3 discloses a device for the cultivation of adherent cells, which is operated as a one-way system in a continuous process. A special feature of this device is the homogenization of the culture medium in the reactor vessel by means of a horizontal and a vertical, cushion-shaped pump element, each of which is operated by compressed air. Appropriate flow distributors ensure uniform mixing. The culture medium is gassed through semi-permeable membrane tubes. However, this device has the disadvantage that it is only designed for the cultivation of adherent cells, but not of stem cells with their special requirements.
    From US4649117A a reactor is known, which can be used as a fermenter for the cultivation of cells, with a particularly shear-free mixing being achieved in that the reactor consists of an inner and an outer chamber and a gentle gas flow centrally from below is initiated. However, this reactor is not suitable for cultivating adherent cells or stem cells, since no corresponding growth areas are provided.
    The object of the present invention is to use the advantages of the known devices, to avoid the disadvantages and to provide a simpler, more flexible solution to the problem of stem cell culture. The microbioreactor module can be used advantageously as a disposable system. A parallel arrangement of several modules in a common or in separate culture rooms enables use as a multimicrobioreactor. In an arrangement as a multi-microbioreactor, the microbioreactor module is particularly suitable for screening and selecting optimal cultivation conditions due to its defined and controllable microenvironment.
    exemplary embodiments
    For a better understanding of the present invention, this will be explained in more detail with reference to the exemplary embodiments illustrated in the following figures. The same parts are provided with the same reference numbers and the same component designations.
    The associated drawings show in Figure 1 a schematic representation of the microbioreactor module, a schematic representation of the microbioreactor module, wherein the cultivation of the stem cells takes place in a cultivation container (1). FIG. 2 shows a schematic representation of a multimicrobioreactor, in which a plurality of microbioreactor modules are introduced into a cultivation space according to DE 10 2014 001 615.3. Figure 3 shows a schematic representation of a top view of the cover a schematic representation of a top view of the cover (11), in which, in addition to a number of module slots (17) open at the top, there are also various connection options for probes (18) that provide online process control allow at different positions in the reactor vessel (12). FIG. 4 shows a schematic representation of a microbioreactor module, a schematic representation of a microbioreactor module, the thorough mixing of the medium in the reactor vessel (12) being ensured by an arrangement corresponding to a bubble column or a loop reactor. FIG. 5 shows a schematic representation of a scale-up bioreactor containing the modules according to the invention. FIG. 6 shows a schematic representation of a side view of a scale-up bioreactor, a schematic representation of a side view of a scale-up bioreactor, with a gas-permeable air bag being located inside the cultivation container. FIG. 7 shows a schematic representation of the front view and a schematic representation of the front view and the side view of a scale-up bioreactor. Figure 8 is a schematic representation of a scale-up bioreactor, wherein a schematic representation of a scale-up bioreactor, wherein the left figure shows the state without active air supply (overpressure in the reactor), and the right figure shows the state with active air supply, which, among other things, Mimicking blood pressure (systolic and diastolic) can be applied. FIG. 9 shows a schematic representation of a scale-up cultivation unit with a large growth surface.
    A microbioreactor module is shown in FIG. 1, with the cultivation of the stem cells taking place in a cultivation container (1) that imitates the microenvironment of a stem cell niche. The cultivation container (1) of the microbioreactor module consists of a bag made of a semi-permeable natural or synthetic membrane material, so that an exchange of small molecules, such as amino acids or glucose, with the environment is possible, but the cell culture is physically fixed. The cultivation container (1) is fitted with a biocompatible carrier material or scaffold (preferably organic or inorganic polymer materials) and can also be used for co-cultivation with, for example, mesenchymal stem cells by appropriately populated carriers.
    The cultivation container (1) is attached to a gas-permeable support tube (2), preferably made of plastic, which ends at the other end in a plug (3) made of plastic, rubber or silicone. The plug (3) is asymmetrical in thickness. In a preferred embodiment, the gas-permeable support tube (2) is made of an elastic or semi-elastic material.
    A discharge and supply line (4) runs inside the holding tube (2), which enables the inoculation of cells, the supply of bioactive molecules and nutrients, and the removal of samples. In a preferred embodiment, the discharge and supply line (4) is made of an elastic or semi-elastic material.
    The discharge and supply line (4) ends on the top of the plug (3) in a connecting piece (5), which can be arranged with a thread on the plug (3), the connecting piece (5) is used as an adapter for the Attaching, for example, a syringe (6) formed, via which the supply and removal of substances, medium and cells is regulated. The opening of the line (4) in the connecting piece (5) is provided with an elastic sealing material, which allows piercing with the attached syringe (6) and closes when the syringe (6) is removed.
    The support tube (2) is introduced with the plug (3) in a casing (7) which is open towards the bottom. The casing (7) has a total of three openings on the side walls. Two of the openings are at the level of the plug (3), another in the lower third of the casing (7). The three openings include two through-flow openings (8) which are provided with a flap (9) which opens or closes depending on the direction of flow of the external medium. The third opening does not have such a flap and serves as a discharge (10) for waste produced, for example unwanted metabolites. The asymmetrical thickness of the stopper (3) and its nature enable targeted opening or closing of either the through-flow opening (8) or the derivation (10) by turning the stopper (3) in the casing (7). The casing (7) preferably consists of a semi-elastic plastic material.
    The cultured cells can be harvested either by partial removal via the discharge and supply line (4) for a continuous process, or by separating the cultivation container (1) from the holding tube (2).
    The microbioreactor module according to the invention can be used in a large number of cultivation systems. The module is introduced parallel to the respective main flow direction of the cultivation system in order to ensure that the through-flow openings (8) function.
    [0023] FIG. 2 shows a multimicrobioreactor in which several microbioreactor modules are introduced into a cultivation space according to DE 10 2014 001 615.3. In this preferred embodiment, one or more different microbioreactor modules according to the invention are fixed in parallel in a plastic cover (11) in openings provided with seals and introduced into an exchangeable reactor vessel (12). In the reactor vessel (12) filled with a medium, uniform, gentle and shear-free homogenization is achieved by a pump element (13) driven by compressed air and located on the bottom. The flow direction of the homogenization, which is reinforced by the perforated plates acting as flow distributors (14), runs parallel to the alignment of the microbioreactor modules. Bubble-free gassing of the medium in the reactor vessel is made possible by semi-permeable membrane hoses (15). Temperature control can be achieved by placing the reactor vessel (12) in a heating element (16), the heating element (16) preferably only covering the lower end of the reactor vessel (12) so that a minimal vertical temperature gradient is created inside the reactor vessel (12). .
    The growth conditions in the individual microbioreactor modules can be individually adjusted. The composition of nutrients in the individual cultivation containers (1), which may be equipped with different carrier materials and cell cultures, can be adjusted by separate discharge and supply lines (4) of the individual modules. The length of the microbioreactor modules can vary and thus a different immersion depth in the medium in the reactor vessel (12) can be achieved. As a result, the growth conditions in terms of temperature (corresponding to the vertical temperature gradient) and pressure (corresponding to the hydrostatic pressure) can be measured with the probe and individualized.
    shows a top view of the cover (11), in which there are also various connection options for probes (18) in addition to an upwardly open number of module slots (17), which provide online process control at various positions in the reactor vessel (12) enable.
    The number of individual microbioreactor modules in a single reactor vessel (12) is only limited by the size of the reactor vessel, with the volume of a single module also being able to vary from the microliter to milliliter scale.
    shows another possible embodiment of a reactor vessel for an application of the microbioreactor module, wherein the mixing of the medium in the reactor vessel (12) is ensured by an arrangement corresponding to a bubble column or a loop reactor. An upwardly open plate with small, grid-like openings serves as a bubble generator (19), through which pulsating compressed air flows via a compressed air supply (20). The cover (11) is connected to the reactor vessel (12) at the seals (24) and corresponds to the arrangement from FIGS. 1-3, but additionally contains a pressure relief valve (23). The bubbles generated by the bubble generator (19) cause a mixing flow in the vertical direction, which flows upwards within an inner reactor shell (21) which is open at the top and bottom and is fixed to the reactor vessel by means of holders (22). In the space between the inner reactor shell (21) and the reactor vessel (12), a corresponding counterflow occurs. Excess air is routed to the outside through the pressure relief valve (23). The opening and closing of the pressure relief valve (23) is accompanied by the pulsating supply of compressed air, so that the increased pressure when air is supplied is replaced by a relaxation phase. One or more microbioreactor modules are placed in the cover (11) parallel to the flow direction in analogy to the preferred embodiment shown in FIGS. 1-3.
    In a preferred embodiment, the microbioreactor module is used to screen a suitable microenvironment for the respective cell type to be cultivated. Here, the composition and concentration of various growth factors, the presence of factors of the extracellular matrix, the dissolved oxygen concentration, the pH value, the osmolarity and the continuous supply of nutrients as well as the removal of metabolites are optimized.
    Another preferred embodiment is the scale-up version of the bioreactor, which enables the desired cell type to be cultivated under optimized conditions on a large scale. Figs. 5-9 show schematic representations of a scale-up bioreactor. The scale-up bioreactor contains one or more modules according to the invention, which contain a larger volume and thus enable a high cell yield under optimized, controlled and reproducible conditions. The three-dimensional culture in a microbioreactor allows for rapid implementation in production and a simple scale-up process. Furthermore, the controllable parameters during cultivation enable a simple and gentle cell harvest.
    In a further embodiment, the scale-up bioreactor enables continuous fermentation due to the controllable cultivation conditions. In one embodiment, the scale-up bioreactor has a bubble generator (19) above and below the cultivation tank (1).
    A further advantage of the microbioreactor module in general, and of the scale-up bioreactor in particular, is the possibility of varying the pressure in the cultivation container in order to, for example, control the blood pressure, which varies in the body of an individual with systole and diastole. (blood pressure 120/60 mmHg, ie 1160/60 mbar). In the microbioreactor module, this is made possible by the compressed air supply (20) and the bubble generator (19). In the scale-up bioreactor there is a gas-permeable air bag (25) in the cultivation container, which simulates the pulsating physical properties of the pulsating blood in the cultivation container by changing the volume and pressure in the air bag. This goes hand in hand with the homogenization within the cultivation tank. Furthermore, the mass transfer as well as the oxygen exchange is promoted by the pressure gradient generated on the surface of the cultivation container. The air bag also has the advantage that it enables the gas exchange of carbon dioxide (CO2) and ammonium (NH4).
    Reference List
    1: cultivation container 2: holding tube 3: stopper 4: discharge and supply line 5: connector 6: syringe 7: jacket 8: flow opening 9: flap 10: discharge line 11: cover 12: reactor vessel 13: pump element 14: flow distributor 15: membrane hose 16: heating element 17: module slots 18: probes 19: bubble generator 20: compressed air supply 21: inner reactor shell 22: bracket 23: pressure relief valve 24: gasket 25: gas-permeable air bag
    权利要求:
    Claims (9)
    [1]
    A microbioreactor module comprising a cultivation vessel (1), a support tube (2), an asymmetric plug (3), and a shroud (7), the microbioreactor module being characterized in that• the cultivation container (1) has an outer shell made of a semipermeable membrane material, which encloses a biocompatible carrier material or a framework made of a biocompatible carrier material,• the cultivation container (1) is attached to one end of the holding tube (2) which encloses a discharge and supply line (4) which allows samples to be taken and cells or bioactive molecules and nutrients to be supplied to the cultivation container (1) allows• the asymmetric plug (3) is arranged at another end of the support tube (2) and has an asymmetric thickness, the asymmetric plug (3) being arranged to protrude into the interior of the microbioreactor module, through the microbioreactor module Seal contact with the casing (7) to the outside, and depending on the positioning of the asymmetrical plug (3) to open or close openings in the wall of the casing (7).
    [2]
    2. Microbioreactor module according to claim 1, characterized in that the outer shell of the cultivation container (1) consists of a bag made of a semipermeable organic or inorganic membrane material with an exclusion size of 1-50 kDa, for example a dialysis tube material.
    [3]
    3. microbioreactor module according to any one of claims 1 or 2, characterized in that the carrier material in the cultivation container (1).(i) natural or synthetic polymers,(ii) inorganic materials, or(iii) a combination thereof, wherein the carrier material comprises collagen, elastin, fibrin, alginate, silk, glycoaminoglucan, hyaluronic acid, chitosan, cellulose, fucoidan or silaffin.
    [4]
    4. Microbioreactor module according to one of claims 1 to 3, characterized in that the holding tube (2), the discharge and supply line (4) and the casing (7) consist of an elastic, gas-permeable material, e.g. silicone and that the casing (7) has a plurality of flow openings (8) which can be closed by flaps (9).
    [5]
    5. microbioreactor module according to any one of claims 1 to 4, characterized in that the cultivation container (1) includes a gas-permeable air bag.
    [6]
    6. Multimicrobioreactor, consisting of several microbioreactor modules according to one of claims 1-5, which are optionally asbilden in different lengths and associated immersion depths, and which are arranged in parallel in a common cultivation space, the growth conditions in the individual microbioreactor modules are individually adaptable by the composition of nutrients and supply in the cultivation container (1).
    [7]
    7. Multimicrobioreactor according to claim 6, characterized in that one or more microbioreactor modules are fixed in a common plastic cover (11) in which connection options for measuring probes (18) and optionally pressure relief valves (23) are also provided.
    [8]
    8. Multimicrobioreactor according to one of Claims 6 or 7, characterized in that the common cultivation space is a reactor vessel (12) filled with medium, in which homogenization is carried out by a compressed air-operated pump element (13) in combination with a flow distributor (14) or by a Bubble generator (19) is ensured, the bubble generator (19) being set up to conduct pulsating compressed air upwards through a perforated plate functioning as a flow distributor (14).
    [9]
    9. Multimicrobioreactor according to claim 8, characterized in that for flow circulation of the medium inside the reactor vessel (12), an inner reactor shell (21) open at the top and bottom is fixed to the reactor vessel (12) via brackets (22).
    类似技术:
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    同族专利:
    公开号 | 公开日
    KR20190063478A|2019-06-07|
    DE102016119391B3|2018-01-18|
    CN109906267A|2019-06-18|
    JP2019534703A|2019-12-05|
    WO2018069169A1|2018-04-19|
    US20200040292A1|2020-02-06|
    KR20210152027A|2021-12-14|
    KR102338639B1|2021-12-13|
    SG11201903231WA|2019-05-30|
    引用文献:
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    法律状态:
    2019-04-30| PK| Correction|Free format text: BERICHTIGUNG INHABER |
    2021-11-15| PL| Patent ceased|
    优先权:
    申请号 | 申请日 | 专利标题
    DE102016119391.7A|DE102016119391B3|2016-10-12|2016-10-12|Microbioreactor module|
    PCT/EP2017/075442|WO2018069169A1|2016-10-12|2017-10-06|Microbioreactor module|
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