KR20230104064A - Bioreactors and related apparatuses - Google Patents

Abstract

A bioreactor that provides compressive load and electrical stimulation is disclosed. The disclosed bioreactor includes: a cell culture plate comprising one or more wells; a base frame equipped with a fixture for fixing cell culture plates of different sizes; and a stimulus transmission unit transmitting physical stimulus to the biomaterial in the well. The bioreactor is freely mobile in use, sterilizable, versatile, easy to assemble and operate, and easy to clean.

Description

Bioreactors and related apparatus {Bioreactors and related apparatuses}

The present invention relates to a bioreactor and a related device, and more particularly, to a bioreactor using mechanical stimulation such as pressure, torsion, and expansion and/or physical stimulation such as electrical stimulation. More specifically, the present invention relates to a multi-purpose bioreactor for individually or simultaneously delivering various physical stimuli to cells including hydrogels, bioinks, tissue engineering scaffolds, cells, cell masses or tissue engineering structures, and related devices. will be.

A bioreactor refers to a system capable of artificially reproducing a biochemical reaction process such as physical stimulation, decomposition, synthesis, or chemical conversion of a substance, which is occurring in the body of a living organism. Bioreactors can be used for biomimetics, operating conditions and environmental factors, which can be used for cell culture, 3D tissue growth, bioink, in vitro tissue regeneration and tissue engineering, stem cell differentiation, cell therapy, 3D bioprinting, medical devices, and regeneration. It plays an important role in fields such as medicine.

In the field of in vitro tissue regeneration and tissue engineering regenerative medicine, it has been demonstrated that physical stimulation helps cells grow and proliferate to form active tissues within or without a scaffold, and in some cases It helps to differentiate or re-differentiate a robust cell line into a specific lineage. In particular, by providing physical stimulation that mimics the biological environment for tissue regeneration, inducing the promotion of in vitro tissue regeneration, significantly shortening the period of regenerating human tissue compared to the period of regenerating human tissue under static conditions, and also regenerating It is a useful technique for inducing tissues into tissues similar to human tissues. Among these physical stimuli, mechanical and electrical stimulation are techniques that are widely used by clinicians as well as researchers.

The physical stimulation applied to the patient includes mechanical stimulation in which hydrostatic pressure generated as the compression and restoration of cartilage tissue by body load is repeated, such as knee cartilage, is applied to cartilage; electric stimulation of the chewing function of teeth in dentistry and the osseous-nervous tissue of the spine; torsional stimulation of tendon tissue caused by exercise; Induction of differentiation and proliferation of stem cells by electric/compressive stimulation; There is repetitive physical stimulation of expansion and contraction movements that occur in the cardiovascular system and cardiovascular muscle tissue. In order to provide in vitro physical stimulation that can mimic the physical stimulation of the patient's body, researchers use various models of bioreactors (bioreactors) in a laboratory environment to induce differentiation of cells and regeneration of tissues or specific organs. is using physical stimulation.

For example, the hydrostatic load generated in cartilage is primarily a mechanical stimulus for the purpose of generating variable, repetitive pressure on a scaffold seeded with cells, a hydrogel material, or direct (in the absence of a scaffold) uniform pressure on the cells. As a study, a study on improving the physical properties of human cartilage tissue regeneration using a biaxial load mechanical stimulation bioreactor system (2017, Scientific Report), stress of dynamic mechanical stimulation using a membrane micro device array capable of transforming mechanical-biological stimulation It has been reported that mesenchymal stem cell substrate production promotion of mesenchymal stem cells (stromal cells) through strain (Sci Adv, 2021). Body stimulation (pulsating pressure, shear force, torsional force, etc.) during tissue regeneration (Birth Defects Res C Embryo Today, 2013, 99(3): 203-222), blood vessel growth factor, cartilage growth factor, nerve Neurons, chondrocytes, osteocytes, etc. A study on the application of cell differentiation technology using physical stimulation, such as a method for acquiring differentiated cells, and applying various modes of electrical stimulation such as direct current and square wave stimulation to control the level of calcium ions through the calcium ion channel in the cell wall. Studies on the regulation of cell activity, skin cell protein enzyme (Akt) polarization, and endothelial cell elongation have been reported (J Biological Eng, 2015).

Korean Patent Registration No. 10-2040690 discloses an automated bioreactor for precise control of cell proliferation and differentiation using gas and medium replacement, that is, pressure.

Korean Patent Registration No. 10-1814440 is an invention related to tissue regeneration using centrifugal force, and discloses a method for producing bead-shaped cartilage tissue by coagulating costal chondrocytes and culturing the cells in 96 wells.

As such, in the prior art, only one of physical stimuli, such as mechanical stimulation such as direct pressure, twisting, and expansion, and electrical stimulation, was used in a bioreactor on a tissue engineering structure (support), tissue regeneration using a combination of the above physical stimuli at the same time, Cell control and the like are not disclosed.

Accordingly, the present inventors applied pressure difference (differential pressure) applied to a specific microscopic structure of a tissue engineering scaffold, which was not attempted in the prior art, compression applied to a defective part of a complex shape such as bone-cartilage composite tissue, physical stimulation that simulates twist, Electrical stimulation, physical stimulation-electric stimulation can be applied simultaneously, physical stimulation by multiaxial extension, twisting, etc. is applied to the same device, that is, by directly modifying, modifying, and utilizing the in vitro cell culture plate to achieve the above physical stimulation. This led to the development of a bioreactor that delivers By using the bioreactor of the present invention, various physical stimuli can be applied to cells at the same time, and different physical stimuli can be applied depending on the location, so that cells can be trained and differentiated more closely to the in vivo system, and biomimetic environment. tissue can be regenerated.

Hydrostatic loading is primarily used to create uniform pressure directly on cell-loaded scaffolds or gel materials or cells in the absence of scaffolds. Direct compression pressure on tissue engineering scaffolds has been used in some bioreactors, but differential pressure on a particular construct has never been attempted using the same device, such as a cell culture plate.

On the other hand, conventional cell culture well plates and petridish do not provide electrical or mechanical stimulation by themselves, and culture cells in a static state or shakers or polyurethane It is forcibly stimulated from the outside by using a material with elasticity. In addition, there is no case where compressive load, electrical stimulation, and dynamic load are simultaneously applied to the defects of various shapes configured differently according to the complex shape and height, and the perfusion system for circulation of the cell culture medium and removal of the used medium does not exist. Accordingly, in the conventional cell culture well plate, the used medium is replaced with a fresh cell culture medium manually by the researcher at regular time intervals, and the automation of the programmed cell culture timing and fresh medium exchange It has no function, and there are problems such as not being able to maintain a constant freshness of the cell culture medium for the entire period of cell culture, as well as the problem of not being able to exchange (circulate / recycle) the used medium.

Therefore, the present inventors have proposed such mechanical and electrical physical stimulation, stimulation applicable to complex shapes, cyclic stimulation of torsion, cyclic stimulation of expansion-contraction, simultaneous provision of electrical stimulation-physical stimulation, and replacement of in vitro cell culture medium. It has led to the development of cell culture plate assemblies specifically designed to solve the problem of medium delivery and recycling.

An object of the present invention is to provide an in vitro bioreactor that mechanically and electrically stimulates cells, including hydrogels, bioinks (hydrogels containing cells), tissue engineering scaffolds, cells, cell masses, scaffolds, or tissue engineering structures. It is to do.

Another object of the present invention is to provide an in vitro bioreactor that provides physical and electrical stimulation by connecting drives having various shapes and heights to a cover of a cell culture plate and deforming the base plate.

Another object of the present invention is to provide a bioreactor capable of applying differential pressure (different pressures), physical stimulation applicable to various shapes, expansion and compression, torsion stimulation, electrical stimulation, and physical stimulation in combination thereof.

Another object of the present invention is to provide a bioreactor capable of precisely controlling the amount of strain (stress deformation, mechanical physical stimulation) due to applied physical stimuli, and the stress level and stress waveform.

Another object of the present invention is a bioreactor capable of applying electrical physical stimulation capable of providing electrical stimulation in addition to mechanical physical stimulation such as compression, expansion, and twisting. It is to provide a reactor.

Another object of the present invention is to provide cell differentiation and tissue regeneration induction technology and results by applying stimuli such as pressure, extension, twist, and electricity to cell systems such as growth factors, drugs, and cells contained in cell culture wells. it is for

Another object of the present invention is compressive load, electrical stimulation, differential pressure and expansion, mechanical mechanical stimulation, As a mechanical control system that can be created by imitating mechanical torque, etc., it is possible to implement compression and expansion application modes, and can control the amount of mechanical physical stimulation deformation from a very low level to a high level. It is to provide a bioreactor capable of applying any type of pressure, such as square, sawtooth, etc., to cells and a support containing cells according to a set program.

Another object of the present invention is to obtain an in vitro regenerated tissue by providing physical stimulation to cells included in a tissue regeneration scaffold by providing physical stimulation of expansion and twisting force to a Petri dish plate.

Another object of the present invention is to provide a bioreactor in which electrical stimulation can be transmitted to cells through a provided channel, and all stimulations can be applied to cells, tissue engineering scaffolds, gels, etc. for a specific period of time or periodically.

Another object of the present invention is a bioreactor capable of delivering electrical stimulation to cells on the sample surface (1-dimensional), inside the sample bulk (3-dimensional), well surface or dish surface by delivering electrical stimulation to an electrically conductive sample. is to provide

Another object of the present invention is to provide a miniaturized bioreactor in which all components are included in a single main body (case).

Another object of the present invention is to provide a bioreactor that is operated with a power supply that has a low risk of heat generation and is easy to replace.

Another object of the present invention is to provide a bioreactor plate compatible with all commercial cell culture plates used for in vitro cell culture and custom cell culture plates manufactured by methods such as 3D printing.

Another object of the present invention is to provide a bioreactor that can be freely moved, can be sterilized, and can be easily placed inside an incubator.

Another object of the present invention is to provide a technique for inducing attachment, proliferation and differentiation, re-differentiation, de-differentiation, and extracellular matrix production of (stem) cells using a combination of various physical and biostimuli-physical stimuli, and to provide results. it is for

Another object of the present invention is to induce tissue regeneration in a scaffold containing cells using various physical stimuli to obtain a tissue regeneration product (film, particle, cell aggregate, etc.) to which various physical stimuli have been applied, At this time, the obtained products (eg, film-type regenerated tissue) are stacked and mixed to obtain a composite regenerated tissue composed of one or more tissues, heterogeneous tissue, or a composite (eg, cell mass mixture).

Another object of the present invention is to provide biomaterial composites containing cells, cell masses, cells, growth factors, exosomes, nano-micro particles, bioactive substances, drugs, etc., hydrogels, bioinks, tissue engineering scaffolds, and cells. It is to provide a cell culture plate well (cell culture plate well) and a cell culture Petri dish comprising.

A bioreactor according to one aspect of the present invention includes a cell culture plate including one or more wells; A base frame having a fixing table for fixing cell culture plates of different sizes; and a stimulus transmission unit transmitting a physical stimulus to the biomaterial in the well.

The stimulation delivery unit. and a cover plate detachably coupled to the reciprocating device and having one or more replaceable heads, the replaceable heads periodically compressing the biomaterial in the well according to the reciprocating motion of the reciprocating device.

A plurality of replaceable heads may be coupled to the cover plate, and at least one of the plurality of replaceable heads may include an electrode for applying electrical stimulation to the biomaterial in the well.

A plurality of replaceable heads are coupled to the cover plate, and the plurality of replaceable heads include heads having cross sections of various shapes, section heads including surfaces having different heights due to steps, and a surface applying pressure to a horizontal surface. It includes at least one of a uniform head made of a material, a differential head including a surface having a gradually different height to apply differential pressure, and a head provided with electrodes to simultaneously apply compression and electrical stimulation.

The surface of the head can be adjusted in various ways, such as a lattice, a curve, a height difference, a step shape, a pyramid, etc., as needed.

The holder is configured to hold cell culture plates of various specifications, including 1, 2, 4, 6, 12, 24, 48 or 96 well plates or Petri dishes.

The fixing base includes a cross shape in which four straight bars intersect at the center, and a screw hole is provided at an end of the straight bar to which a fixing screw is installed so as to advance or retreat in a threaded manner.

The bioreactor is assembled with a solenoid drive, and the solenoid drive includes a disk-type magnet for applying an impact load to the biomaterial.

The stimulation transmission unit includes an expansion applying mechanism coupled to a lower end of the reciprocating mechanism.

The expansion applying mechanism is rotatably installed at the lower end of the reciprocating mechanism and a pair of holders holding both ends of the sample in order to provide periodic expansion in the uniaxial direction to the sample immersed in the culture medium in the well. includes a pair of guiders connected to the pair of holders, and the pair of guiders convert the up and down reciprocating motion of the reciprocating mechanism into uniaxial expansion and contraction motion of the sample.

The extension applying mechanism is rotatable at the lower end of the reciprocating mechanism and two pairs of holders holding the four ends of the sample in order to provide periodic expansion in the biaxial direction to the sample immersed in the culture medium in the well. It is installed and includes two pairs of guiders connected to the two pairs of holders at the bottom, and the two pairs of guiders convert the up and down reciprocation of the reciprocating mechanism into biaxial expansion and contraction movements of the sample.

The stimulation transmission unit includes a twist applying mechanism coupled to a lower end of the reciprocating mechanism, and the twist applying mechanism includes a fixed holder that can contact the cell culture plate and fixes one side of the sample, and the reciprocating movement. It includes a movable holder that is connected to a driver coupled in a crank manner to the lower end of the mechanism, each rotates by the reciprocating motion of the reciprocating mechanism, fixes the other side of the sample, and applies twist to the sample.

The stimulation transmission unit further includes a power supply device, a controller, and a timer relay to control a period of periodically stimulating the biomaterial and a stimulation application cycle.

The cell culture plate includes a plurality of wells, a supply unit for supplying recirculated or fresh medium from the outside, an inlet formed on the inner upper part of each well through which medium is injected, an outlet formed on the inner lower part of each well, and a recirculated medium. Includes a drain through which the medium is discharged to the outside.

The supply unit and the drain are connected to a supply line and a discharge line connected to the circulation pump and the medium storage tank to complete the medium circulation system.

The cell culture plate includes a perfusion path sequentially connecting the plurality of wells between the supply unit and the drain.

The cell culture plate includes a plurality of supply parts and a plurality of drains, and the cell culture plate includes an upper flow path connecting inlets of two neighboring wells between two neighboring wells and two neighboring wells between two neighboring wells. It includes a lower flow path connecting between outlets of the plurality of supply units, each of which is connected to the upper flow path, and each of the plurality of drains is connected to the lower flow path.

An electrode for applying electrical stimulation may be positioned in each of the wells.

The bioreactor further includes a cage for holding the electrode within the well.

The inlet or the outlet is covered with a stopper structure in the form of a screen, semi-permeable membrane, woven or non-woven material, micro/nano fiber sheet or gel.

A cell culture plate assembly is provided according to another aspect of the present invention, wherein the cell culture plate assembly includes: a cell culture plate comprising one or more wells; and at least one recirculation system coupled with the cell culture plate for recirculation or replacement of media in the wells, the cell culture plate comprising: One or more wells, a supply unit for supplying recirculated or fresh medium from the outside, an inlet formed on the inner upper part of each well through which the medium is injected, an outlet formed on the inner lower part of each well, and a medium to be recycled to the outside. Including the discharged drain.

The medium circulation system includes a medium storage tank, a supply line connected to the supply unit,

A discharge line connected to the drain, and a circulation pump circulating the medium to supply the medium in the medium reservoir to the cell culture plate through the supply line and recover the medium in the cell culture plate to the medium reservoir through the discharge line. include

The cell culture plate includes a perfusion path sequentially connecting the one or more wells between the supply unit and the drain. The cell culture plate includes one or more supply parts and one or more drains, and the cell culture plate has an upper flow path connecting inlets of two neighboring wells between two neighboring wells and a neighboring one between two neighboring wells. It includes a lower passage connecting the outlets of the two wells, each of the one or more supply parts is connected to the upper passage, and each of the one or more drains is connected to the lower passage.

Since the present invention is freely movable in use, sterilizable, versatile, easy to assemble and operate, easy to clean, and can easily apply and control mechanical and electrical stimuli individually and simultaneously, the user In research on tissue regeneration through the application of physical stimulation, multiple physical stimulation effects can be applied at once to effectively promote tissue regeneration and stem cell differentiation that require physical stimulation. More specifically, by simultaneously/comprehensively providing various physical stimuli to the existing tissue engineering scaffold (cell-bioactive material-tissue engineering scaffold) and stem cells that provided growth factors, drugs, exosomes, and cells, in vitro tissue It can promote regeneration and stem cell proliferation and differentiation. Body load such as cartilage, meniscus, spine, tendon, ligament, muscle, etc., physical muscle exercise by exercise, muscle exercise such as heart/vascular by blood circulation, or physical stimulation such as esophagus where peristalsis of food is applied It can provide an in vitro tissue regeneration system and technology that mimics, and has the expected effect of inducing cell attachment, proliferation, differentiation and tissue regeneration by providing physical stimulation to cells, stem cells, etc. using various physical stimuli. . For example, 1) the expected effect of changing the behavior of nerve cells in nerve tissue and maxillofacial tissue by simultaneously applying electrical stimulation or electrical stimulation and differential pressure, and 2) the expected effect of differentiating into nerve cells and regenerating nerve tissue. 3) In the case of tendons and ligaments, since they do not have blood vessels, it is difficult to restore them once they are broken. Therefore, using torsion/extension stimulation, the tissue regeneration effect of tendons and ligaments is expected. 4) In the case of cartilage, cartilage regeneration is promoted using body weight. Expected effect of tissue regeneration by stimulating biological behavior, 5) Expected effect of inducing muscle tissue regeneration by providing stimuli generated from beating motion and peristaltic motion in the case of blood vessels, heart, esophagus, or airway, 6) Well plate It has an expected effect of simultaneously applying different physical stimuli to each well or one well constituting .

The technology that has been available to date includes a mechanical or electrical method, a direct compression method and a hydrostatic pressure method as the mechanical method, and a compression or expansion application mode in the direct pressure method. On the other hand, in the present invention, it is possible to provide compression and twist in a uniaxial or biaxial direction. Until now, available technologies have not been able to provide adjustable and precise control over the deformation range and differential pressure generation for the same sample, but the present invention provides adjustable and precise control over the deformation range and differential pressure generation for the same sample. It is possible.

The present invention has the advantage of being versatile (precise, adjustable and positive control over compression or expansion of the sample), portable, sterile, and can easily fit into the interior of any cell culture incubator. And the present invention can deliver programmed mechanical and electrical stimuli to the sample using a single setup. The number of samples, type and intensity of stimulation can also be changed as desired.

In addition, the present invention makes it easy to individually and simultaneously control mechanical and electrical stimuli in a series of samples, and allows researchers to apply various physical stimuli to tissue engineering scaffolds at once in multiple wells of one plate. Help.

The present invention has an effect of promoting tissue regeneration by providing physical stimuli under various conditions to bioprinting or cell-containing scaffolds.

The present invention can promote in vitro tissue regeneration such as cartilage and dental by applying a compressive load to the tissue engineering scaffold.

In the present invention, multi-layered tissue (eg, cartilage) can be regenerated by providing a compressive load to a multi-layered biodegradable scaffold containing cells and providing different physical stimuli for each layer.

According to the present invention, in vitro tissue regeneration of tendons and ligaments can be promoted by periodically providing torsional physical stimulation.

The present invention can promote in vitro tissue regeneration by providing various and complex physical stimuli by freely replacing and providing cover heads of various shapes.

The present invention can promote tissue regeneration, such as the esophagus, blood vessel airway, etc., by periodically providing an expansion stimulus to the tissue engineering scaffold.

The present invention can shorten the in vitro tissue regeneration time and reduce the risk of contamination that may occur during tissue regeneration by promoting tissue regeneration by periodically providing physical-electrical stimulation to the tissue engineering scaffold.

The present invention can be applied to induce differentiation of stem cells in vitro because compression, expansion, twisting, and electrical stimulation can be provided.

In a future perspective, using the bioreactor configuration of the present invention, regenerated cells in single or multiple steps with various cell lineages differentiated from potent cells (or stem cells) to produce functionally active multi-layered tissues or organs. In vitro tissue can be applied to tissue engineering scaffolds for tissue engineering and regenerative medicine (eg in vivo ).

The cell culture plate assembly presented in the present invention can simultaneously apply mechanical and electrical stimulation along with programmed perfusion through a counter electrode fixed at an exact position. Furthermore, the present invention is portable, sterilizable, can easily fit inside any incubator, and can be used without causing internal temperature rise in delivering continuous supply and recirculation of fluid or cell culture medium. Additionally, a single setup can be used to deliver programmed perfusion to the sample and serve as an appropriately placed counter electrode for tissue engineering configurations, and the flow rate, duration, and type of circulation can also be varied as desired. The cell culture plate assembly of the present invention can not only be used by itself or in combination with a commonly used cell culture well plate cover, but also can be combined with the disclosed elements in Korean Patent Application No. 10-2021-0192557 to provide electrical and physical stimulation. At the same time as zooming, the medium can be automatically replaced/recirculated during the cell culture process.

The cell culture plate assembly of the present invention and the bioreactor including the same can simultaneously apply mechanical and electrical stimuli together with programmed perfusion of a counter electrode fixed at an accurate position, and deliver continuous supply and recirculation of fluid or cell culture medium. It is portable, sterile, fits easily inside any incubator, uses a single setup to deliver programmed perfusion to samples, and has the effect of being properly positioned for tissue engineering configurations. The effects of the present invention are mentioned above, but are not limited to the effects. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

The effects of the present invention are mentioned above, but are not limited to the effects. It should be understood that the effects of the present invention include all effects that can be inferred from the following description.

Figure 1 shows a bioreactor assembled with a specially designed cover plate to apply pressure and electrical stimulation as mechanical stimulation to the tissue regeneration scaffold.
Figure 2 shows a bioreactor assembled with an expansion application mechanism specially designed for the expansion mode.
FIG. 3 is a perspective view from above of a universal base frame, a reciprocating piston coupled thereto, and a specially designed cover plate, which are part of the bioreactor shown in FIG. 1;
4 is a perspective view of the structure shown in FIG. 3 viewed from below.
5 shows a state in which various heads are coupled to holes of the specially designed cover plate shown in FIGS. 3 and 4 .
6 is a view showing various examples of a head coupled to a specially designed cover plate.
FIG. 7 shows a bioreactor and accessories assembled with a solenoid drive mounted on a cell culture plate to which a specially designed cover plate is coupled.
8 is for explaining an experimental example of the present invention.
9 to 14 are photographs showing the results over time after applying electrical stimulation or mechanical stimulation to the sample.
15 and 16 are diagrams for explaining a bioreactor including a uniaxial extension applying mechanism.
17 is a view for explaining a bioreactor including a biaxial extension applying mechanism.
18 and 19 are views for explaining a bioreactor including a torsion applying mechanism.
20 exemplarily shows various types of bioreactors according to the present invention.
21 and 25 are diagrams for explaining an embodiment of a cell culture plate for a bioreactor in which a perfusion mechanism for recirculating and replacing cell culture medium in programmed multi-wells is integrated.
26 to 28 are diagrams for explaining another embodiment of a cell culture plate for a bioreactor in which a perfusion mechanism for recycling and replacement of cell culture medium in programmed multi-wells is integrated.
29 to 32 show an example applied to the above-described bioreactor using the cell culture plate 1000 described above, wherein cells are loaded on the alginate gel and then a compressive load of a certain thickness is applied to the cell inside the alginate gel. These are photographs showing the results of observing changes in cell viability, cell proliferation, and production of extracellular matrix such as collagen and GAG according to changes in their behavior.

Hereinafter, with reference to the drawings, look at the embodiments of the present invention in detail.

1 shows a bioreactor assembled with a specially designed cover plate 200 to apply pressure and electrical stimulation as mechanical stimulation to a tissue regeneration scaffold, and FIG. 2 shows a specially designed expansion application mechanism 300 for an expansion mode. Shows the assembled bioreactor.

The bioreactor includes a universal base frame 100 as shown in FIGS. 1 and 2 .

The bioreactor also includes a stimulation delivery unit for delivering mechanical stimulation, electrical stimulation or mechanical stimulation and/or electrical stimulation to the cells or any sample in the cell culture plate.

In the mode of applying pressure or electrical stimulation to the sample with various head(s), the stimulation delivery unit, as shown in FIG. 1, includes a motor 10, an adjustable cam 20, a reciprocating piston 30, Together with the power supply 40, the controller 50, the timer relay 60, and the power source 70 for the timer relay, with the help of these, it operates to reciprocate up and down periodically, and the sample is subjected to pressure stimulation and/or Alternatively, it may include a cover plate 200 applying electrical stimulation.

In the extended application mode for applying the expanded stimulus to the sample, the stimulus transmission unit, as shown in FIG. 2, includes a motor 10, an adjustable cam 20, a reciprocating piston 30, and a power supply 40 ), the controller 50, the timer relay 60, and the power supply 70 for the timer relay, and with the help of them, an expansion application mechanism 300 for applying an expansion force to the sample. The expansion application mechanism 300 may be a uniaxial direction expansion application mechanism, or alternatively, a biaxial expansion application mechanism.

In the torsion application mode for applying the torsional stimulation to the sample, the stimulation transmission unit includes a motor 10, an adjustable cam 20, a reciprocating piston 30, a power supply 40, a controller 50, In addition to the timer relay 60 and the power supply 70 for the timer relay, a twist applying mechanism for applying twist to the sample with the help of these may be included.

Switching between the foregoing modes can be accomplished by replacing the instrument assembled on the same reciprocating piston 30 and changing the cell culture plates 2000 and 3000 detachably secured by the universal base frame 100. can The cell culture plate 2000 shown in FIG. 1 is preferably a 6, 12, 24, 48 or 96 well plate formed in an approximately rectangular or square shape and containing a plurality of wells, and the cell culture plate shown in FIG. 2 ( 3000) is preferably an approximately circular Petri dish containing one well.

Referring to FIGS. 1 and 2 , the stimulation transmission unit provides mechanical stimulation and / Alternatively, electrical stimulation may be delivered to the sample according to a program.

The controller 50, more specifically, the microcontroller 50 may control the RPM of the motor 10. A power source 70 for operating the timer relay 60 is additionally provided.

3 is a perspective view from above of a universal base frame, a reciprocating piston coupled thereto, and a specially designed cover plate, which are part of the bioreactor shown in FIG. 1, and FIG. 4 is a perspective view of the structure shown in FIG. 3 viewed from below. 5 shows a state in which various heads are coupled to holes of the specially designed cover plate shown in FIGS. 3 and 4, and FIG. 6 is a view showing various examples of heads coupled to the specially designed cover plate.

Referring to Figures 3 to 6, the universal base frame 100 is 1, 6, 12, 24, 48 or 96 well plates or various cell culture plates including petri dishes (2000 or 3000; see Figs. 1 and 2). ) Includes a fixing table 120 capable of fixing. The fixing table 120 may have a cross shape in which four straight bars intersect at the center, and at the end of the straight bar, a jaw 122 having a screw hole 123 in which a fixing screw is installed to advance or retreat in a threaded manner is provided. do. A screw hole 124 into which an adjustment screw is installed may be formed at the center of the holder 120 to adjust the height of the cell culture plate (2000 or 3000; see FIGS. 1 and 2).

The universal base frame 100 or the cell culture plate may serve as a ground for electrical signals. The universal base frame 100 includes a platform capable of receiving cell culture plates from 5 mm to 300 mm or other vessels for cell culture/tissue engineering purposes with dimensions from 5 mm to 300 mm in all three directions. The overall dimension of the universal base frame 100 is less than 500 mm.

In addition, the universal base frame 100 includes an upper support 140 located at a predetermined interval above the fixing table 120, and a guide 142 in the form of a hollow cylinder is formed on the upper support 140. As a part of the press actuator, the reciprocating piston 30 is installed to be reciprocating up and down along the guide 142 . A guide 142 guides the press actuator to an intended sample placed within a cell culture well (or vessel or container).

A connection portion 32 connected to an adjustable drive cam 20 (see FIGS. 1 and 2) connected to the motor shaft is formed at an upper end of the reciprocating piston 30. A motor support 170 for holding the motor 10 (see FIGS. 1 and 2) is formed on one side of the universal base frame 100. Although not shown, the universal base frame 100 may further include one or more supports for fixing a controller, a timer, a display, an electrical signal input generator, and a power supply.

As mentioned above, the bioreactor includes a stimulation delivery unit for delivering mechanical stimulation, electrical stimulation or mechanical stimulation and/or electrical stimulation to the cells or any sample in the cell culture plate.

1, 3 to 6, the stimulation transmission unit cooperates with the power supply 40, the controller 50, the motor 10, and the adjustable drive cam 20 as a reciprocating driving means. , a specially designed cover plate 200 for delivering both mechanical and electrical stimuli to the sample.

The specially designed cover plate 200 is connected to the adjustable drive cam 20 installed on the motor shaft and is controlled by “+” so that the weight of the sample is not loaded. In addition, the specially designed cover plate 200 may provide electrical stimulation to the sample from the power supply 40 .

The specially designed cover plate 200 has detachable pressure/extension heads 202 at the bottom to compress or expand the sample. The head 210 may be connected to the power supply 40, and electrical stimulation may be applied to the sample through the head 210. The number, shape and location of the replaceable heads 210 can be varied according to the researcher's requirements. As will be described below, the replaceable head 210 includes a section head (a) including surfaces having different heights by steps to apply pressure with different heights, and surfaces having gradually different heights from the central portion to the edge portion. A differential head (b) capable of applying a differential pressure by applying a differential pressure, a uniform head (c) having a horizontal surface to apply pressure to apply a uniform pressure, and an electrode having the shape of the above-described head so as to apply electrical stimulation. There may be one head (d, e, f).

The same universal base frame 100 can be used with a microcontroller 50 and power supply 40 to deliver a square wave or impulse pressure using a solenoid drive. Also, the universal base frame 100 can be used as a ground for electrical signals.

The entire configuration of the bioreactor is portable, sterilizable, and can be easily secured inside the incubator by clips, clamps, or other suitable fixing means that operate without vibration.

As mentioned above, the universal base frame 100 includes one or more guides 142 and input electrical signal grounds for guiding compression actuators to intended samples located in wells or vessels of a cell culture plate, and having an overall dimension of less than 500 mm. It may include features.

In one embodiment, at least one adjustable drive cam 20 is installed on the shaft of the motor 10 to enable screw adjustment, and displacement control of 0 to 20 mm is possible using a guide screw for position adjustment. . Using drive screws with finer or finer threads, positioning accuracy can be less than 0.01mm. Alternatively, position control may be implemented using other mechanisms such as slots, worms, worm-worm wheels, pins, and racks. The adjustable drive cam 20 may further include an adjustable connection that can adjust the distance between the cam positions to drive the drive head using screws or other types of adjustments.

In addition to the screw-type adjustable cam 20 described above, a controlled mechanical stimulus may be applied to the sample and the cells located inside the cell culture plate by using an arbitrary reciprocating driving mechanism such as a solenoid or a piezoelectric actuator.

Referring to Figures 1 and 2, at least one reciprocating piston 30 may be coupled with a cam or other drive mechanism to achieve reciprocating motion. In addition, the lower end of the reciprocating piston 30 is threaded at the bottom to connect to a cover plate 200 specially designed for cell culture plates, an expansion application mechanism 300, or many other specially designed compression or expansion heads or electrodes, or both. includes A specially designed cover plate 200, an extended application mechanism 300, or other compression head, which may or may not have electrodes, is attached to the lower end of the reciprocating piston 30, so that living cells, hydrogels, biomolecules , mechanical stimulation, electrical stimulation, or both mechanical and electrical stimulation can be applied to a subject having a metal, non-metal, alloy, polymer, or combination thereof for the purpose of growing functionally active tissue regeneration in vitro. Any mechanism such as a slot, clip, etc. may be used to attach the reciprocating piston to the pressure/expansion head, electrode or both.

1, 3 to 6, the cover plate 200 for a cell culture plate (specifically, a 6, 12, 24, 48 or 96 well plate) includes a reciprocating piston 30 that reciprocates. attached to the bottom of The cover plate 200 is a metal or non-metal for the purpose of growing an object in a cell culture plate, that is, living cells, hydrogels, biomolecules, or functionally active tissue regeneration in vitro by pressing pressure electrodes or electrical stimulation or both. , one or more holes 202 for attaching replaceable heads 210 for application to an object having an alloy, polymer, or combination thereof. These holes 202 are located in the center of standard cell culture wells of cell culture plates 2000 . A screw may be provided in the hole 202 to hold the replaceable head 210 and provide an electrical pulse.

5 and 6 show several compression heads, ie, interchangeable compression heads 210, for applying various pressures on the tissue regeneration scaffold. The replaceable compression head 210 can be assembled by fitting into the hole 202 of the cover plate 200 . The interchangeable compression heads 210 have dimensions from 1 mm to 300 mm in one, two or three directions. The interchangeable compression heads 210 may be cylindrical, cuboidal, cuboidal, pyramidal, conical, or in the shape of a living cell, hydrogel, biomolecule, metal for the purpose of growing functionally active tissue regeneration in vitro, It has any three-dimensional shape specially designed to be applied to objects having non-metals, alloys, polymers, or combinations thereof. The replaceable heads 210 have any shape specifically designed to apply differential compression pressure to an object. The replaceable heads 210 have a partially filled, solid or hollow shape to give the intended stimulus to the object. The replaceable heads 210 have one or more electrodes for transmitting electrical signals to the object.

As well shown in FIG. 6, the replaceable head 210 includes a section head (a) including surfaces having different heights by steps to apply pressure with different heights, and a surface for applying pressure to apply uniform pressure is a horizontal plane A uniform head (b) made of, a differential head (c) capable of applying differential pressure including a surface whose height gradually varies from the center part to the edge part, and a head having an electrode 211 to apply compression and electrical stimulation ( d, e, f), and may include a head applying electrical stimulation. When manufacturing the head, the head surface may be manufactured by adjusting various shapes such as a lattice, a curve, a height difference, a stair shape, and a star.

The electrode 211 is for transmitting various electrical signals, and for example, one or more holes may be drilled in the head 210 to provide the electrode 211 or a wire connected thereto. The lower surface of the head 210 can be designed in various ways and its height adjusted to provide differential pressure or differential load to the object.

The bioreactor includes a universal base frame capable of fixing various cell culture plates and combined with various stimulation delivery units.

As briefly mentioned above, FIGS. 3 and 4 show a specially designed cover plate 200 corresponding to a 24-well cell culture plate as part of a 24-well stimulation delivery unit and a universal equipped with a mechanism for reciprocating the cover plate 200. The base frame 100 is shown as an example.

As briefly mentioned above, the universal base frame 100 includes a fixing base 120 and an upper support 140 positioned above the fixing base 120 at a predetermined interval. A guide 142 in the form of a hollow cylinder is formed on the upper support 140 . A reciprocating piston 30 connected to an adjustable driver cam, which is a reciprocating driving mechanism, is installed to be capable of reciprocating up and down along the guide 142 . A guide 142 guides the press actuator to an intended sample placed within a cell culture well (or vessel or container). As the motor rotates, the adjustable drive cam connected to the motor shaft reciprocates the reciprocating piston 30.

The fixing table 140 may have a cross shape in which four straight bars intersect at the center, and at the end of the straight bar, a jaw 122 having a screw hole 123 in which a fixing screw is installed to advance or retreat in a threaded manner is provided. do. A screw hole 124 into which an adjusting screw is installed may be formed at the center of the holder 120 to adjust the height of the cell culture plate.

7 shows a bioreactor and accessories assembled by mounting a solenoid drive 10' on a cell culture plate 2000 to which a specially designed cover plate 200 is coupled. In the solenoid driver 10', disk-type magnets with a weight of 3 grams (weight/magnet) are used to apply an impact load to the sample. The magnet number is used to impart the proper load to the sample. In addition, the bioreactor includes a bioreactor power source connected to the solenoid driver 10', that is, a power supply device 40', and a power supply device 40' connected to the power supply device 40' and a solenoid driver ( 10'), a timer relay 60', and a power supply 70' for timer relay.

[Experimental example]

For cell culture, 4 g of the Na salt of alginic acid was dissolved in 100 mL of deionized water with continuous stirring at 400 rpm on a magnetic stirrer overnight. A solution is prepared by adding 100 mM CaCl ₂ to phosphate buffered saline (PBS) (1.1 g in 100 ml PBS), followed by constant stirring for 1 hour. For alginate gel preparation, 100 μl of CaCl ₂ solution per 1 ml of alginate solution was used.

8 shows a bioreactor setup operating in a CO ₂ incubator. 8 (a) is electrical stimulation, (b) is mechanical stimulation, (c) is gel compression according to cyclic load test, and (d) is various examples of compression heads used during the study (mechanical stimulation from the third to the left). 8 (e) shows the gel in a 24-well plate after cyclic mechanical compression for 1 day (an impression of the compression head is shown).

For mechanical stimulation and electrical stimulation studies, two bioreactors were set in an incubator and used (Fig. 8 (a) and (b)). 2 mm at 25 ºC for cylindrical gel samples (18.5 mm in height and 10 mm in diameter, similar in volume to a 24-well cell culture plate) using a texture analyzer analysis instrument (Stable Micro Systems model, TA.XT plus, Surrey, UK). Cyclic compression was applied at a rate of /min to evaluate the mechanical properties of the samples. Figure 4 (d) shows the shapes of various heads and ends to which a 2.3 mm compressive load is applied, which provides a 1 N load to the sample for compression study, and the bioink containing cells under the condition corresponding to a 12.7 kPa pressure load. (cell-embedded hydrogel) behavior and cellular response were evaluated (consistent with available literature). When the first three compression heads were applied, the bioink showed a periodic recovery history even under cyclical compression loads. In one set of samples, the sample height was adjusted to provide a 1 mm compressive load (low pressure samples). The driving cam frequency of the bioreactor with mechanical compressive loading was adjusted to 1 Hz using 2.3 mm. In Figure 1e, by observing that the shape of the hydrogel according to compression was observed, it was observed that the shape of the compression head, which indicates that the bioreactor was applied to the sample, was present in the gel.

In order to understand the amount of voltage required, the experimental conditions were selected based on literature references, and when 5V (d.c) was applied by day 1 of in vitro cell culture, it was observed that it decreased to 2V by day 7. A programmable timer relay was calibrated to deliver a 5 second pulse with a 5 second pause. Cell culture studies used a combination cell culture medium (MEM alpha solution + FBS (10%) + ABAM (1%) (Sigma Aldrich)). Since ionic conductivity is the main contributor to electrical conductivity in this experiment, the amount of current passing through the gel sample was measured using the culture medium. For the short electrostimulation head, 2ml solution (height 13.4 mm, 10 mm diameter cylindrical sample) was used, and for the long electrostimulation head, 1 ml solution (height 6.7 mm, diameter 10 mm cylindrical sample) was used. After immersion in the medium for 1 hour, the resistance of the gel was observed to be 105 ± 5 kΩ and 85 ± 5 kΩ, respectively. Therefore, the amperage was observed to be around 50μA (thick sample) and 60μA (thin sample) for 5V and 20μA (thick sample) and 25μA (thin sample) for 2V power supply.

For cell culture studies, osteoblast cell lines (Passage 13, MC3T3-E1 cell line, Young Science Inc., Korea) derived from rat skulls were subcultured in 24-well plates. α-MEM (Minimum essential media) (Sigma Aldrich, USA) containing 10% fetal bovine serum (Gibco Korea, Korea) and penicillin-streptomycin (100 unit/mL) was added to each well and incubated for 24 hours in 5% CO ₂ . incubated for hours. At 37 °C, the 24 well plate was connected to a mechanical and electrical bioreactor setup as shown in Figure 1. All alginate solutions were mixed with 1 million/ml cells and cross-linked with CaCl ₂ solution (100 μl/ml). Control samples had no electrical or mechanical stimulation applied.

In vitro cell culture studies were performed for up to 7 days, and samples were observed on days 1, 3 and 7. For live/dead staining, the LIVE/DEAD Viability/Cytotoxicity Kit (Invitrogen, USA) was used. Intracellular esterase activity by combining green fluorescent calcein-AM staining with red fluorescent ethidium homodimer-1 staining was used to determine the integrity of the plasma membrane. Loss confirmed. Images of live and dead cells were taken after adding 1.2 μL of 2 mM ethidium homodimer-1 (EthD-1) and 0.3 μL of 4 mM calcein AM to 600 μL of phosphate buffered saline (PBS). After adding the above two formulations, the plate was incubated for 30 minutes in the dark. Live and dead cells were imaged using different filters on a fluorescence microscope (Leica DMLB, Germany) and merged using the LAS-X Leica microsystem software (Fig. 9-A1, A2).

[Electrical pulse application experiment]

Figure 9 shows the cell behavior inside the bioink after 1 day of electrical stimulation, (a) control, (b) thin sample, (c) thick sample, (d) thick sample with a probe inserted at a distance of 7 mm in cross section. indicate The scale bar is 1 mm.

Referring to FIG. 9, 1 day after cutting the 5v pulse, it was observed that the sample (in this case, a thin sample was used) was deteriorated and damaged due to the differential head using a sharp probe, and all cells died. Set was excluded from the experiment. The rest of the sample set showed an improvement in the area of cell proliferation compared to the control. However, by observing the growth of the thin sample and the failure of the differential head experiment, the experiment was conducted by reducing the voltage to 2v until the end of the experiment.

10 shows cell behavior inside bioink 3 days after applying electric stimulation, (a) control, (b) thin sample, (c) thick sample, (d) thick sample with a probe inserted at a distance of 7 mm in cross section. indicates The scale bar is 1 mm.

Referring to FIG. 10 , after 3 days, insufficient cell growth was observed. A decrease in the number of cells was observed, although slight growth could be observed in thick samples with a current of less than 20 μA.

11 shows the cell behavior inside the bioink after 7 days of applying electric stimulation, (a) control, (b) thin sample, (c) thick sample, (d) thick sample with a probe inserted at a distance of 7 mm from the cross section. indicates The scale bar is 1 mm.

Referring to FIG. 11 , on the 7th day, it was observed that the cells inside the thin gel sample died and the sample also lost its own structure, and cell proliferation was observed only in the thicker sample.

From this experiment, it can be concluded that electric pulses have a significant effect on cell growth and proliferation. The probe distance must be adequate to deliver a current of 20 μA or less, and a shorter distance could damage cells and gels due to high current. It has been observed that high voltages and currents are useful for short periods of time, such as one day. It was analyzed that using a lower voltage and current for a longer time was justified, and the pulse period was adjusted to reduce the electrical stress of the sample.

[Mechanical pressure application test]

12 shows changes in the behavior of bioink according to the application of mechanical (compressive) load on day 1. (a) is a control group, (b) is a load applied with a section head, and (c) is a uniform head (uniform). head), (d) shows a differential head, (e) shows a less compressed (1 mm) sample. The scale bar is 1 mm.

12 shows cell proliferation after 1 day, and it was observed that there was a distinct growth difference between the high pressure region and the low pressure region of the section head (Fig. 12(b)). Cells in the high pressure region were observed to grow and proliferate faster than those in the low pressure region. However, higher pressures can lead to cell death, as in differential heads where more cell death is observed in the high pressure region. It was observed that uniform pressure led to more uniform cell proliferation over the entire sample area.

13 shows changes in the behavior of bioink according to the application of mechanical (compressive) load on day 3. (a) is a control group, (b) is a load applied with a section head, and (c) is a uniform head (uniform). head), (d) shows a differential head, (e) shows a less compressed (1 mm) sample. The scale bar is 1 mm.

13 shows the results of cell culture after 3 days. The high pressure region of the section head shows a much broader cytoplasm than the low pressure region. A uniform head delivering a cyclic compressive load of 1N shows very good cell proliferation. However, it was observed that a low load was not very effective as shown in FIG. 13(f). The differential compression head showed a tendency for cell damage in the higher section of the compression load region.

14 shows changes in the behavior of bioink according to the application of mechanical (compression) load on day 7. (a) is a control group, (b) is a load applied with a section head, and (c) is a uniform head (uniform). head), (d) shows a differential head, (e) shows a less compressed (1 mm) sample. The scale bar is 1 mm.

Results found after 7 days confirmed that pressure has a significant effect on promoting growth and proliferation of cells encased in the gel matrix. Pressed areas in all heads show much better cell proliferation as observed in FIG. 14 .

This study led to the conclusion that compressive load stimulation can be used to enhance cell growth. A study to optimize the compressive load load cycle and its magnitude was subsequently conducted.

Hereinafter, a bioreactor used in a 1-axis or 2-axis expansion application mode and a torsion application mode will be described. Even in the above various modes, the mechanism for reciprocating motion including the universal base frame, motor, adjustable cam, and reciprocating piston can be used as it is or almost as it is already described above.

Referring to Figures 2, 15 and 16, an expansion application mechanism 300 as an attachment to the reciprocating piston 30 is fabric, sheet, fabric (woven, non-woven, knitted, braided and others), mesh, hollow For tubes, cylinders, and other structures, at least two or more holders 320 and 320 may be included to generate expansion force by holding ends at two or more points. The holder 320 can accommodate a structure for periodic longitudinal extension of 10 mm to 200 mm in length, 0.001 mm to 50 mm in thickness and 0.001 mm to 50 mm in width, and may be partially or completely submerged in a liquid such as a cell culture medium. can

15 and 16 are diagrams for explaining a bioreactor for uniaxial expansion including the aforementioned universal frame 100, the Petri plate 3000, and the expansion applying mechanism 300 for uniaxial expansion.

As shown in FIGS. 15 and 16 , the extension applying mechanism 300 for uniaxial extension is coupled to the lower end of the reciprocating piston 30 . The expansion applying mechanism 300 for uniaxial expansion is a pair of holders 340 holding both ends of the sample in order to provide periodic expansion in the uniaxial direction to the sample submerged in the culture medium in the Petri plate 3000, which is a cell culture plate. , 340), and a pair of holder guiders (or sliders; 320, 320) rotatably installed at the lower end of the reciprocating piston 30 and connected to the pair of holders 340 and 340 at the lower end. do. A pair of guiders (or sliders) 320 and 30 for the holder can convert the up and down reciprocating motion of the reciprocating piston 30 into expansion and contraction motions of the sample, that is, the structure. And, the surface of the holder 340 in contact with the sample may have a flat surface, a nano/micro structure, an inclination, and the like. A pair of holders (340, 340) is within the cell culture plate, more specifically, the petri dish (3000). Holding both ends of samples including fibers, sheets, fabrics (woven fabrics, non-woven fabrics, knitted fabrics, braids and others), meshes, hollow tubes, cylinders and other structures, the reciprocating motion of the reciprocating piston 30 and the resulting By moving the pair of guiders 320 and 320, the sample may be periodically stretched in one axis direction. The pair of guiders 320 and 320 push the pair of holders 340 and 340 outward or pull them inward by the up and down reciprocation of the reciprocating piston 30, and thus, the pair of holders 340 and 340 ), the sample fixed at both ends is periodically subjected to an extension force in the uniaxial direction.

FIG. 17 is a view for explaining a bioreactor for biaxial expansion including the aforementioned universal frame 100, a Petri plate 3000, and an expansion applying mechanism 300' for biaxial expansion. As shown in FIG. 17, an extension applying mechanism 300' for two-axis extension is coupled to the lower end of the reciprocating piston 30. The expansion application mechanism 300' for biaxial expansion provides periodic stretching in two axial directions, that is, in the X-axis direction and in the Y-axis direction orthogonal thereto, to the sample at least partially submerged in the culture medium in the Petri plate 3000. In order to do this, two pairs of holders 340 'holding the left and right four ends of the sample and two pairs of holders 340' rotatably installed at the bottom of the reciprocating piston 30 and connected to the two pairs of holders 340' at the bottom It includes a guider (or slider; 320') for the holder. The two pairs of guiders (or sliders) 320' for holders allow the up and down reciprocation of the reciprocating piston 30 to be converted into biaxial expansion and contraction movements of the sample, that is, the structure. Also, the surface of the holder 340' that contacts the sample may have a flat surface, a nano/micro structure, or an inclined surface. The two pairs of holders 340' are cell culture plates, more specifically, within the Petri dish 3000. Reciprocating motion of the reciprocating piston 30 and Due to the resulting movement of the pair of guiders 320', the sample can be periodically stretched in the biaxial direction. By the up and down reciprocation of the reciprocating piston 30, the two pairs of guiders 320' push the two pairs of holders 340' outward or pull them in, and thus, by the two pairs of holders 340' The sample fixed at the four ends of the left and right sides is periodically applied with an expansion force in two axial directions intersecting each other.

18 and 19 are views for explaining a bioreactor for applying twist including the universal frame 100, the Petri plate 3000, and the twist applying mechanism 400 described above.

Referring to FIGS. 18 and 19 , a torsional application mechanism 400 as an attachment to the reciprocating piston 30 may be fabric, sheet, fabric (woven, non-woven, knitted, braided and others), mesh, hollow tube, cylinder and at least two holders 420 and 440 to hold both ends and apply twist to the sample, which may have a different structure.

Here, at least one holder 420 of the two holders is a fixed holder 420 and the other at least one is connected by a hinge 432 to a driver 430 connected to the reciprocating piston 30 and a crank type to reciprocate It is a movable holder 440 that rotates each rotation according to the reciprocating motion of the motion piston 30. The fixing holder 420 may fix one side of the sample S while being fixed to the cell culture well plate 3000 or the Petri dish 3000 . The up and down reciprocating motion of the reciprocating piston 30 is converted into a periodic torsional motion of the sample S by the driver 430 and the moving holder 440 to apply a torsional force. The fixed holder 420 is provided with a first fixing pin 422 for fixing one side of the sample (S), and the movable holder 440 has a second fixing pin 442 for fixing the other side of the sample (S). ) may be provided. 19 shows an action of applying twist to the sample (S) by rotating the movable holder 440 from Position 1 to Position 2 by the reciprocating piston 30 and the driver 430 connected in a cranked manner thereto.

The holder can accommodate regular or irregularly shaped structures ranging from 10 mm to 200 mm in length, 0.001 mm to 50 mm in thickness, and 0.001 mm to 50 mm in width, which are partially or completely immersed in a liquid such as a cell culture medium.

FIG. 20 shows various forms of various bioreactors by way of example. In FIG. 20 (a), a drive cover accommodating drive elements that drive a stimulus transmission unit such as a motor, a power supply, and a controller is integrated into a universal base frame. 20(b) shows a bioreactor including a cell culture plate made of a rectangular single container or well, and FIG. 20(c) shows mechanical stimulation (compression, expansion) by a stimulus delivery unit. or torsion) and/or a cover covering a cell culture plate to which electrical stimulation is applied.

Meanwhile, the aforementioned bioreactor and/or elements constituting the bioreactor may be metal, non-metal or plastic.

Additionally, the bioreactor and/or components comprising the bioreactor may be sterilized by one or more of the conventional sterilization protocols in a cell culture laboratory.

To protect such a sterile state, the sterile bioreactor and/or elements constituting the bioreactor may be covered with an appropriate cover.

Process materials to which mechanical stimulation and electrical stimulation are applied are living cells, cell masses, gels, nano or micro particles, biomolecules, hydrogels, polymers, crosslinking agents, tissue engineering scaffolds, 3D printed structures, 3D bioprinted structures, and elastomers. and mixtures thereof.

A bioreactor comprising the foregoing components may be assembled and installed inside a laboratory incubator by means of clips, clamps, or other suitable fixing means that operate without vibration, intended, without an external power supply, intended, controlled and It can be operated for programmed stimulus application.

In one embodiment, for the purpose of in vitro active tissue growth, mechanical and electrical stimulation is applied to structures or cell delivery systems having multi-component materials including polymers, gels, nano and micro particles, biomolecules, bioinks and living cells. can be applied For purposes other than the above, mechanical and electrical stimuli may be applied to bioinks or structures having multi-component materials including polymers, gels, nano and microparticles, biomolecules and living cells.

In one embodiment, the molding material of the aforementioned components may be metal, non-metal, alloy, plastic or composite.

In one embodiment, the aforementioned components may be sterilized through one or more conventional sterilization protocols used in cell culture laboratories.

In one embodiment, the process material for applying mechanical and electrical stimulation is a living cell, cell mass, gel, nano or micro particle, biomolecule, polymer, crosslinking agent, bioink, tissue engineering scaffold, 3D printed structure, 3D bioprinted structure , elastomers, and mixtures thereof.

In one embodiment, an electrical stimulus may be delivered to the sample and applied to cells in or on the sample, cells on the surface of a well plate or on the surface of a petri dish.

In one embodiment, the sample may have electrical conductivity, and more specifically, it may be an electrically conductive gel containing electrically conductive carbon nanotubes in a hydrogel.

In one embodiment, the entire assembly of the above-mentioned components and process materials can be housed inside a laboratory incubator, capable of intended control, and operated for programmed stimulation application without an external power supply. can

In one embodiment, the present invention provides a structure having multi-component materials including polymers, gels, nano- and micro-particles, biomolecules, cell masses and living cells for the purpose of regenerating functionally active tissue in an incubator (bio ink or support) may include a method of applying mechanical and electrical stimuli.

In one embodiment, the present invention provides multi-component materials, including polymers, gels, nano- and micro-particles, biomolecules, cell masses and living cells, for purposes other than regenerating functionally active tissue in an incubator. It may include a method of applying mechanical and electrical stimulation to a structure (bioink or scaffold) having a structure.

In one embodiment, the present invention can apply materials and shapes capable of physical stimulation to biomaterials, hydrogels, bioinks, cell carriers, and tissue engineering scaffolds.

In one embodiment, the cell culture plate of the present invention includes plates having various wells (6, 12, 24, 48, 96 wells) and cell culture dishes (5 cm, 10 cm, culture dishes manufactured in other sizes, etc.) can include

In one embodiment, the power supply may be a 0 to 9 volt battery that has a low risk of overheating and is easy to replace.

In one embodiment, a shock load may be applied to the sample using a disc-shaped magnet weighing 3 g per solenoid driven attachment to the bioreactor and a 24-well cell culture plate. The number of the disk-shaped magnets can be adjusted to deliver an appropriate load, such as a body load, to the sample. Electrical stimulation can use a 0 to 9 volt battery, voltage can be controlled by rpm/power controller, pulse, duration, etc. can be controlled by a program controlled by a time relay. there is.

In one embodiment, the entire configuration can be miniaturized by including all in one case.

In one embodiment, the bioreactor induces tissue regeneration in a cell-containing scaffold using physical stimulation to obtain tissue regeneration samples (film, particle, etc.) to which various conditions of physical stimulation are applied, and each sample (eg, film) obtained at this time A single regenerated tissue can be obtained by stacking the regenerated tissue). For example, cartilage is composed of several layers (epidermal layer, middle layer, lower layer, bone-cartilage interface, etc.), and when physically stimulated layers are stacked, a composite layer of cartilage tissue can be obtained, which is used to replace lost cartilage tissue. can be applied

Hereinafter, a cell culture plate assembly for a bioreactor that can be applied to various applications of the bioreactor described above or to the various bioreactors described above will be described.

The cell culture plate assembly of the present invention described below may be applied to the aforementioned bioreactor(s), or may be used alone or in combination with other types of bioreactors.

21 is a schematic view showing an example in which the cell culture plate assembly according to an embodiment of the present invention is applied to the bioreactor described above, and FIG. 22 is a view for explaining the cell culture plate in more detail.

The cell culture plate assembly for a bioreactor according to this embodiment has a biodegradable tissue engineering structure (support), provides mechanical stimulation and electrical stimulation to the tissue engineering structure (support), and the tissue engineering structure includes cells or cells It is a system that can culture cells while applying physical stimulation to biomaterials, tissue engineering structures, or cells without containing

In one embodiment, a cell culture plate assembly for a bioreactor includes a cell culture plate 1000, wherein the cell culture plate 1000 includes a plurality of wells or containers 1110 and a supply unit 1120 into which recirculated or fresh medium is introduced. ), a supply inlet 1130 covered with a screen on the inside of the well, a supply control cap 1140, a cage 1150 for fixing the electrode, an electrode fixing channel 1160, and a medium to prevent backflow It includes a siphon (1170) adjustable to the water, a discharge port (1180) at the bottom covered with a screen or membrane, and a drain (1190) through which the waste medium solution or the medium solution to be recycled is discharged.

The medium provided through the supply line moves the medium from the well 1110 to the other well 1110 through the siphon 1170 and moves out of the cell culture plate 1100 through the medium discharge line L1, thereby discharging the used medium. sent to the medium reservoir (1001). The medium in the medium storage tank 1001 is transferred to the medium supply line L2 through the circulation pump 1002 and fresh medium is continuously passed through the cell culture plate 1000 to induce circulation of the medium.

According to one embodiment, the cell culture plate assembly includes a power supply 1003 for supplying power to the circulation pump 1002, a controller 1004 for controlling the circulation pump 1002 and the power supply 1003, It may include a timer relay 1005, a power supply 1006 for the timer relay, and a display.

23 (a) and (b) are perspective views showing a height adjusting cap 1140 for a medium supply line and a siphon 1170 for a medium delivery or medium drain line, respectively.

24 shows an inner cage (or nest; 1150) for fixing electrodes provided in a cell culture plate. The inner cage 1150 for fixing the electrode is divided into an inlet, which is an empty space in the upper membrane structure, and an outlet made of a screen or membrane. An inner cage (nest) for electrical stimulation may be located at a distance of 0 mm to 100 mm from the inner surface of the well 1110 (see FIG. 21 ). The cage 1150 is provided with an electrode 1152, and the electrode 1152 may be, for example, stainless steel or platinum.

25 (a) and (b) are for explaining a current supply model for a 3-dimensional sample and a current supply model for a 2-dimensional sample by an electrode fixed by an electrode fixing channel.

The current supply model shown in (a) of FIG. 25 generates a lateral flow of current across the sample. The + electrode is provided from the top head (not shown) and the sample is sandwiched between the bottom metal electrode and the top head electrode and provides current along the entire volume of the sample (following the cross-section).

The current supply model shown in (b) of FIG. 25 generates a longitudinal flow of current in a two-dimensional sample. A sample is sandwiched between the + electrode and the - electrode, and the electrons provide current along the sample in the form of a film, for example, in the longitudinal direction of the sample.

Referring back to FIGS. 21 and 22 , the cell culture plate 1000 includes one or more wells or containers 1110 having a cylindrical, cubic, cuboidal, conical, spherical, regular or irregular shape for cell culture or tissue engineering. do.

In one embodiment, the cell culture plate 1000 is 5 to 300 mm, and the dimensions of each well 1110 are 1 mm to 300 mm in one, two or three dimensions.

In one embodiment, the well or container 1100 includes at least one perfusion pathway, channel or pipe.

In one embodiment, the cell culture plate 1000 may be used alone or may be used in combination with a cover.

In one embodiment, the cover may be a generally commercially available cover or a cover capable of applying mechanical stimulation such as compressive load stimulation or electrical stimulation separately or simultaneously, that is, the cover plate described above.

In one embodiment, the pump 1002 for perfusion or circulation uses a medium inlet tubing connected to a vacuum pump to introduce fresh medium, or to discharge used medium to the outside using tubing after a suction pump. .

In one embodiment, pump 1002 for perfusion or circulation may operate on 0 to 50V direct current.

In one embodiment, the inner cage 1150 for fixing the electrode is divided into an inlet, which is an empty space in the upper membrane structure, and an outlet made of a screen or membrane.

In one embodiment, the siphon 1170 is for transferring and discharging the medium between the wells 1110 and the wells 1110 and can prevent the medium used in the wells 1110 from flowing backward.

In one embodiment, the siphon 1170 is positioned higher than the medium to prevent the medium from moving from well 1110 to well 1110, and a suction pump is used to aspirate the medium in the lower position to the next well. is injected into and discharged from

In one embodiment, when electrical stimulation is delivered across the tissue engineering structure, at least one electrode fixed at a specific location in the cell culture container or cell culture well (and electrical connection is connected to the tissue engineering structure while electrical pulses are delivered). It acts as a counter electrode or ground electrode.

In one embodiment, the present invention includes two or more electrodes and electrical connections fixed at specific locations in a well or container to act as electrode pairs while delivering longitudinal or two-dimensional electrode stimulation to a tissue engineered construct.

In one embodiment, the at least one perfusion pathway attached to the circulation pump comprises a regular or irregularly shaped channel or pipe having a diameter of 0 to 20 mm and a length of 0 to 1000 mm.

In one embodiment, at least one pipe or channel of the well or container 1110 has a fluid adjustable or non-adjustable opening.

In one embodiment, the well or container 1110 has at least one opening for a supply or discharge line of a pipe or channel.

In one embodiment, there may be more than one opening of channels for each well through the supply or discharge line.

In one embodiment, the openings of the channels in well 1110 are covered in one or two dimensions with a 0.001 mm to 1 mm perforated screen.

In one embodiment, the openings of the channels in the well 1110 may be covered with a semipermeable membrane, a woven or non-woven material, a micro/nano fiber sheet, or a gel-type stopper structure in addition to the screen.

In one embodiment, the cell culture plate 1000 is cylindrical, cuboidal, conical, spherical, or regular for cell culture or tissue engineering purposes for securing electrodes inside the wells 1110. It contains one well or container with an irregular shape.

In one embodiment, the inner cage 1150 for electrical stimulation is located at a distance of 0 mm to 100 mm from the inside of the well 1110.

In one embodiment, the cell culture plate 1000 has at least one electrical connection to fixed or movable electrodes that can act as a ground or counter electrode for electrical stimulation in a transverse direction.

In one embodiment, the cell culture plate 1000 has at least two or more electrical connection parts for fixed or movable electrodes that can serve as electrodes for giving electrical stimulation in a longitudinal direction.

In one embodiment, the cell culture plate 1000 has a length, width and height of 1 mm to 300 mm in one, two or three dimensions.

In one embodiment, the cell culture plate 1000 has one or more drains for draining fluid.

In one embodiment, the channel opening of the well 1110 is covered with a screen having pores having a size of 0.001 mm to 1 mm in one dimension or two dimensions.

In one embodiment, the opening of the channel is a structure covered with a structure other than a semi-permeable membrane, a non-woven or woven structure, a micro/nano porous sheet, a non-porous sheet or a gel-type structure for the purpose of tissue engineering. have

In one embodiment, the connection between the channel and the circulation pump 1002 may or may not use a separate storage tank and treatment device, and may be unidirectional or bidirectional.

In one embodiment, the supply unit or the input line connection unit of the channel may be connected to a storage tank composed of at least one section to supply a desired fluid to a specific well through a circulation pump.

In one embodiment, the channel's medium delivery or discharge line connection is separate from the supply line and can be connected to a medium reservoir or pump for recirculation.

In one embodiment, the medium delivery or drain line originates from the well and the opening, and at least one or more of the openings is located at a height of 0 to 20 mm from the bottom of the well.

In one embodiment, the media delivery or drain line is designed so that fluid is only expelled when the fluid level rises to an adjustable level or is forced out of the well.

In one embodiment, the material of manufacture of the components making up the cell culture plate may be metal, non-metal or plastic.

In one embodiment, the components constituting the cell culture plate may be sterilized through any one or more of the conventional sterilization protocols used in cell culture laboratories.

In one embodiment, processing materials for mechanical and electrical stimulation include living cells, gels, nano or micro membranes, arrayed fibers, porous or non-porous sheets, nano or micro particles, biomolecules, polymers, crosslinkers, and the like. It may be selected from the group consisting of mixtures of.

In one embodiment, the components and processing materials that make up the cell culture plate can be housed inside a laboratory cell culture apparatus and operate for controlled, programmed perfusion with controlled and programmed stimulation application without an external power supply. can

In one embodiment, a method includes applying a controlled, programmed perfusion to a tissue engineered construct having processing materials for the purpose of growing functionally active tissue in vitro or otherwise. The process material is selected from the group consisting of polymers, gels, micro/nanofibrous membranes, aligned fibers, porous or non-porous sheets, nano and micro particles, biomolecules and living cells.

In one embodiment, including processing materials with polymers, gels, micro/nanofibrous membranes, arrayed fibers, porous or non-porous sheets, nano and micro particles, biomolecules and living cells for functional growth or otherwise. A method of applying mechanical or electrical stimulation in a set, controlled, and programmed perfusion bioreactor to a tissue-engineered construct.

In one embodiment, the bioreactor may be used as a single module, and any combination of such modules may be oriented in a specific orientation for controlled, programmed stimulation application with or without an external power supply.

In one embodiment, stem cells, differentiated cells, tissue engineering products, or regenerated tissues in which stem cell differentiation is induced by physical stimulation may be generated using a bioreactor including a cell culture plate.

On the other hand, in the embodiment described above, it is difficult to control the performance of the siphon to supply the medium to the well plate including a plurality of wells, that is, the cell culture plate, by siphon, and if contamination occurs, 6 may become contaminated at the same time. can The embodiments described below are intended to solve the above problems.

Another embodiment of the present invention will be described with reference to FIGS. 26 and 27 .

Contents not described in the following embodiments are the same as those in the previous embodiment. Also, note that the description of the previous embodiment and the description of the following embodiment may partially overlap. In addition, specific components of the embodiments described below may be included in the previous embodiments, and conversely, specific components described in the previous embodiments may be included in the following embodiments. Furthermore, a component included in one embodiment may be applied to another embodiment instead of a component included in another embodiment within the scope obvious to those skilled in the art.

The cell culture plate assembly comprises a cell culture plate 1000 comprising one or more containers or wells 1110 having a cylindrical, cubic, cuboidal, conical, spherical, or other regular or irregular shape for cell culture or tissue engineering purposes. include In the following description, the term "well" is intended to include a container as well as a well in the general sense.

The cell culture plate 1000 includes a plurality of wells 1110 in a matrix arrangement.

The cell culture plate 1000 has a length, a width, and a width within a range of 5 mm to 300 mm, and three dimensions of each well, that is, a length, a width, and a width are within a range of 1 mm to 300 mm.

According to this embodiment, a plurality of circulation pumps 1002 are independently connected to a cell culture plate 1000 composed of a plurality of wells 1110 (12 in this embodiment), thereby providing a plurality of medium circulation systems. It is configured to supply a medium through. For example, as shown in FIG. 27, in a cell culture plate (or well plate 10000) including 12 wells 1110, 4 medium circulation systems each including 4 circulation pumps 1002 are provided. In operation, each of the three medium circulation systems (A1) among the four medium circulation systems including each circulation pump 1002 is a pair of neighboring wells 1002 and 1002. That is, the medium is circulatively supplied to the two wells, and the other medium circulation system A2 is configured to cyclically supply the medium to the two pairs of wells, that is, four wells.

The circulation pump 1002 for perfusion or circulation can be operated by a 0 to 50 v direct current supply.

Each of the four medium circulation systems corresponding to one cell culture plate may include a medium storage tank 1001, a circulation pump 1002, a controller for fluid circulation control, and a power supply unit. In addition, each badge circulation system may further include a timer relay, a timer relay power supply, and a display. Although not shown, one or more supports may be provided to hold the circulation pump, power supply, controller, timer relay, power supply, and the like.

In the above two embodiments of the cell culture plate assembly, the cell culture plate assembly may include the following configuration.

Cell culture plate 1100 includes one or more perfusion pathways for directing fluid to intended structures located in specific cell culture wells (or vessels) 1110 . A perfusion pathway may include a channel or pipe.

Each well 1110 includes an upper inlet 1130 and a lower outlet 1180, and the lower outlet 1180 is connected to an independent medium circulation system A1 or A2 through a discharge line L1 connected to the drain 1190. Connected, the upper inlet 1130 is connected to an independent medium circulation system through a supply line (L2) connected to the supply unit 1120. Two neighboring upper inlets 1130 and 1130 between two neighboring wells 1110 and 1110 are connected to each other by an upper flow path, and two neighboring lower outlets 1180 and 1180 between two neighboring wells 1110 and 1110 ) are connected to each other by a lower flow path. The supply unit 1120 is connected to the upper flow path and the drain 1190 is connected to the lower flow path, completing the independent medium circulation system A1 for the two wells. The medium supplied to the two wells 1110 through each supply line L2 and the upper inlet by the driving of the circulation pump 1002 passes through the lower inlet of the two wells 1110 and each discharge line L1 to the cells. Moved out of the culture plate 1100, the used medium is sent to the medium reservoir 1001. The medium in the medium storage tank 1001 is delivered to the supply line L2 again through the circulation pump 1002 to deliver fresh medium so that the medium continuously passes through the cell culture plate 1000 to induce circulation of the medium.

In the other medium circulation system, the supply unit 1120 and the drain 1190 are formed between the two neighboring wells 1110 and 1110, respectively, and the two neighboring pairs of wells are adjacent to each other. The two lower outlets 1180 and 1180 adjacent to each other between the two wells are connected to the two upper inlets 1130 and 1130 adjacent to each other between the two adjacent wells by the two upper flow channels, respectively. The supply unit 1120 is connected to the two upper flow channels and the drain 1190 is connected to the two lower flow channels, completing the independent medium circulation system A2 for 4 wells. The medium supplied to the four wells 1110 through the supply lines L2 and L2 and the upper inlet by the circulation pump 1002 is driven by the lower inlet of the corresponding four wells 1110 and the discharge lines L1 and L1. ) through the cell culture plate 1100, and the used medium is sent to the medium reservoir 1001. The medium in the medium storage tank 1001 is again delivered to the supply lines L2 and L2 through the circulation pump 1002 to deliver fresh medium so that the medium continuously passes through the cell culture plate 1000 to induce medium circulation.

In one embodiment, a cell culture plate assembly for a bioreactor includes a cell culture plate 1000, wherein the cell culture plate 1000 includes a plurality of wells or containers 1110 and a plurality of supply units into which recirculated or fresh medium is introduced. 1120, an upper inlet 1130 covered with a screen at the upper inner side of the well, a lower inlet 1180 covered with a screen at the lower inner side of the well, and the upper inlet 1130 adjacent to the two neighboring wells are connected to each other, and the supply unit It may include an upper flow path connected to and a lower flow path connected between two neighboring lower injection ports 1180 of two neighboring wells and connected to a drain.

The cell culture plate 1000 may further include a supply control cap as described in the previous embodiment, a cage for fixing electrodes, and an electrode fixing channel.

The cell culture plate 1000 can also include one or more electrodes and electrical connections that can act as counter (opposite) or ground electrodes while delivering transverse electrical pulses to tissue engineering constructs within the wells. These electrodes and electrical connections can be fixed at specific locations on the cell culture well (or vessel). Transversal electrical pulses may be applied for tissue (eg, nerve tissue, bone tissue) regeneration by electrical stimulation.

Two or more electrodes and electrical connections are secured at specific locations within a cell culture well (or vessel) to act as electrode pairs while delivering longitudinal or two-dimensional electrical pulses to the tissue engineering construct. This is to maintain a constant distance between the tip of the cover drive in the well, the gel sample, and the medium. The overall dimension of the cell culture well plate is 1000 mm on each of the three sides.

The perfusion path to which the circulation pump is coupled includes regular or irregularly shaped channels or pipes having a diameter of 0 to 20 mm and a length of 0 to 1000 mm. The perfusion pathway includes an opening in a channel or pipe within the well for forming a supply line to the well (or vessel), which opening may be flow regulated or non-flow regulated.

The perfusion path to which the circulation pump is coupled includes at least one opening for a discharge drain or delivery or drain line of a pipe or channel of a well (or vessel) for recirculation purposes. The delivery or drain line may have an adjustable siphon arrangement to maintain fluid within the well to a desired height.

There may be one or more separate independent channel openings for individual wells through the supply and drain lines.

The openings of the channels in the well may be covered by a filtering means. Specifically, the openings of the channels in the well may be covered with a screen having a pore size of 0.001 to 1 mm. Alternatively, the channel openings of the wells 1110 may be covered with a structure other than a screen, such as a semi-permeable membrane, a nonwoven or woven structure, a micro/nano fiber sheet, or a gel type plug.

The at least one retaining means for holding the electrode inside the well may be a well shape (or container shape) having a cylindrical, cubic, cuboidal, conical, spherical or other regular or irregular shape for cell culture or tissue engineering purposes. there is. The well shape may or may not be similar to the shape of the well described above. At least one retaining means for holding the electrode inside the well may be located inside the well at a distance of 0 mm to 100 mm from the inner wall of the well.

At least one retaining means for retaining the electrode within the well has one or more electrical connections to the fixed or movable electrode so that the fixed or movable electrode serves as a ground or counter electrode for transverse electrical stimulation.

The means for holding the electrodes within the well may have two or more electrical connections to the fixed or movable electrodes so that the fixed or movable electrodes act as an electrode pair for longitudinal electrical stimulation.

At least one holding means for holding the electrode inside the well has a length, width and height of 1 mm to 300 mm in one, two or three dimensions.

One holding means for holding the electrode inside the well has a drain hole for draining the fluid.

The openings of the channels in the retaining means are covered with a screen having an air gap of 0.001 to 1 mm in one or two dimensions. Alternatively, the openings of the channels in the retaining means may be covered with structures other than screens, such as semi-permeable membranes, non-woven or woven structures, micro/nanofiber sheets, non-porous sheets, or gel-type plugs for tissue engineering purposes.

Channel connections to pumps or circulating pumps may or may not have separate storage tanks and used liquid disposal units in one or both directions.

Separate independent pumps for individual wells or groups of wells may be accommodated in one or more circulation pathways.

The supply or infusion line connections of the aforementioned channels may be connected to a storage tank, which includes one or more sections for supplying fluid intended into a particular well via a circulating pump.

The delivery or drain line connection of the channel is separate from the supply line and can be connected to a used liquid disposal unit or pump for recirculation. The delivery or discharge line originates from a hole formed in the aforementioned well or the aforementioned retaining means. One or more drain holes are located at a height of 0-20 mm from the bottom of the well. The delivery or discharge line from the well is designed so that the fluid level rises to an intended adjustable height or exits the well when forced pumped.

A material for fabricating the parts of the cell culture well plate described above may be metal, non-metal or plastic.

Components of the cell culture well plate described above may be sterilized through one or more of the common sterilization protocols used in cell culture laboratories.

In the cell culture well plate described above, the processing materials to which mechanical and electrical stimulation are applied are micro/nanofiber membranes, aligned fibers, porous or non-porous sheets, nano or micro particles, biomolecules, polymers, crosslinking agents, and mixtures thereof. It may be any of living or non-living things including.

The entire assembly including the cell culture plate described above and the process materials described above can be housed inside a laboratory incubator and controlled perfusion for programmed perfusion and intended control of programmed stimulatory action without an external power supply. can work with

According to one aspect of the present invention, there is provided a medium circulation method using the above-described cell culture plate and the above-described process materials, wherein the medium circulation method is provided for the purpose of growing a functionally active tissue in vitro, a polymer, A mechanical, electrical or any other stimulus is applied to a structure having multi-component materials including gels, micro/nanofibrous membranes, aligned fibers, porous or non-porous sheets, nano and micro particles, biomolecules and living cells.

According to the present invention, a structure having multi-component materials such as polymers, gels, micro/nanofibrous membranes, aligned fibers, porous or non-porous sheets, nanoparticles and microparticles, biomolecules, and living cells for in vitro functionally active tissue A method for growing by applying electrical stimulation to is provided.

In addition to the above objects, a method of applying electrical stimulation to a structure having multi-component materials such as polymers, gels, micro/nanofibrous membranes, aligned fibers, porous or non-porous sheets, nanoparticles and microparticles, biomolecules, and living cells is provided. .

The entire assembly of the foregoing components, including the foregoing cell culture well plates and the like, can be used as a single module, any number of such modules being intentionally controlled and controlled, with programmed stimulation applications with or without an external power supply. It can be arranged in any particular orientation for programmed perfusion.

FIG. 28 shows an example in which the cell culture plate 1000 described above is applied to the bioreactor described above. It is fixed by the fixing screws shown. A stimulus transmission mechanism capable of transmitting electrical and/or mechanical stimuli may be coupled to the reciprocating piston 30 installed to be reciprocating on the upper support 140 of the universal base frame 100 . The stimulus cutting instrument may be a cover plate 200 with interchangeable heads capable of applying pressure and/or electrical stimulation to the sample in the well 1110 (see FIG. 27 ). A case in which various elements for driving the stimulation transmission unit are accommodated may be integrally connected to the universal base frame 100 .

29 shows an example applied to the bioreactor described above using the cell culture plate 1000 described above, and cell behavior changes of cells inside the alginate gel by loading the cells in the alginate gel and then applying a compressive load. This is the result of observing changes in viability, cell proliferation, and production of extracellular matrix such as collagen and GAG. Figure 29 shows the results of cell behavior according to application of static cell culture (static) and bioreactor (dynamic) for 1 day, and Figure 30 shows cell behavior according to application of static cell culture (static) and bioreactor (dynamic) for 3 days. 31 is a measurement result of collagen production according to static and bioreactor (dynamic) application for 4 days, and FIG. 32 is GAG production according to static and bioreactor (dynamic) application for 4 days Indicates the measurement result. Alginate gel was dissolved in 100 mL of deionized water by continuously stirring 4 g of the Na salt of alginic acid on a magnetic stirrer overnight at 400 rpm. A 100 mM CaCl ₂ solution in phosphate buffered saline (PBS) (1.1 g in 100 ml PBS) was prepared with constant stirring for 1 hour. For alginate gel preparation, 100 μl of CaCl ₂ solution was used per 1 ml of alginate solution. The bioreactor compression load was applied periodically, and the amount of collagen and GAG (glycosaminoglycan) was measured while the cell culture was in progress for 4 days. The bioreactor application conditions were 20% strain, load application cycle 0.5 Hz, 2 hours/day, 90 seconds stop and 30 seconds load application cycle. The cells were cultured for 7 days, the density of 250,000 cells/ml, the pre-incubation time before applying the bioreactor was 24 hours, and the total culture period was 7 days. was added well and mixed by hand. The amount of collagen and GAG produced during the cell culture process under static cell culture and the compressive load of the bioreactor were compared and analyzed.

The present invention is not limited by the above-described embodiments and can be variously modified and practiced.

10: motor
20: adjustable cam
30: reciprocating piston
1000, 2000, 3000: cell culture plate
100: universal base frame
200: cover plate
300: 1-axis expansion application mechanism
300': 2-axis expansion approval mechanism
400: torsion applying mechanism

Claims (23)

a cell culture plate comprising one or more wells;
A base frame having a fixing table for fixing cell culture plates of different sizes; and
A bioreactor comprising a stimulus transmission unit that transmits a physical stimulus to the biomaterial in the well. According to claim 1,
The stimulation delivery unit.
a cover plate detachably coupled to the reciprocating mechanism and having one or more replaceable heads;
The replaceable head periodically compresses the biomaterial in the well according to the reciprocating motion of the reciprocating mechanism. According to claim 2,
A plurality of replaceable heads are coupled to the cover plate, and at least one of the plurality of replaceable heads includes an electrode for applying electrical stimulation to the biomaterial in the well. According to claim 2,
A plurality of replaceable heads are coupled to the cover plate,
The plurality of replaceable heads,
A section head including surfaces having different heights by steps;
A uniform head made of a horizontal surface on which a pressure is applied;
A differential head for applying differential pressure including a face whose height gradually changes;
A bioreactor comprising at least one of a head provided with electrodes and simultaneously applying compression and electric stimulation. According to claim 1,
The bioreactor, characterized in that the stationary table is configured to fix cell culture plates of various specifications including 1, 2, 4, 6, 12, 24, 48 or 96 well plates or petri dishes. The fixing unit of claim 1, wherein the fixing unit includes a cross shape in which four straight bars intersect at the center, and an end portion of the straight bar is provided with a screw hole formed with a fixing screw installed so as to be screwed forward or backward. characterized bioreactor. The bioreactor according to claim 2, wherein a solenoid drive is mounted and assembled, and the solenoid drive includes a disk-type magnet for applying an impact load to the biomaterial. According to claim 2.
The bioreactor according to claim 1, wherein the stimulation transmission unit includes an expansion applying mechanism coupled to a lower end of the reciprocating mechanism. According to claim 8.
The expansion applying mechanism is rotatably installed at the lower end of the reciprocating mechanism and a pair of holders holding both ends of the sample in order to provide periodic expansion in the uniaxial direction to the sample immersed in the culture medium in the well. and a pair of guiders connected to the pair of holders, wherein the pair of guiders convert the up and down reciprocating motion of the reciprocating mechanism into uniaxial expansion and contraction motion of the sample Bio reactor. According to claim 8,
The extension applying mechanism is rotatable at the lower end of the reciprocating mechanism and two pairs of holders holding the four ends of the sample in order to provide periodic expansion in the biaxial direction to the sample immersed in the culture medium in the well. It is installed and includes two pairs of guiders connected to the two pairs of holders at the bottom, wherein the two pairs of guiders allow the up and down reciprocation of the reciprocating mechanism to be converted into biaxial expansion and contraction movements of the sample A bioreactor with According to claim 2,
The stimulation transmission unit includes a twist applying mechanism coupled to a lower end of the reciprocating mechanism,
The torsion applying mechanism,
A fixing holder capable of contacting the surface of the cell culture plate and fixing one side of the sample;
Characterized in that it comprises a movable holder connected to a driver coupled in a crank type to the lower end of the reciprocating mechanism, each rotated by the reciprocating motion of the reciprocating mechanism, and fixing the other side of the sample to apply twist to the sample. bioreactor that does. According to claim 1,
The bioreactor further comprises a power supply, a controller, and a timer relay so that the stimulation transmission unit controls a period of periodically stimulating the biomaterial and a stimulation application period. The method of claim 1, wherein the cell culture plate.
one or more wells;
A supply unit for supplying recirculated or fresh medium from the outside;
An inlet formed on the inner upper portion of each well and into which a medium is injected;
An outlet formed on the inner lower part of each well;
A bioreactor comprising a drain through which a medium to be recycled is discharged to the outside. According to claim 13,
The bioreactor, characterized in that the medium circulation system is completed by connecting the supply unit and the drain to a supply line and a discharge line connected to the circulation pump and the medium storage tank. 14. The bioreactor according to claim 13, wherein the cell culture plate having one or more wells includes a perfusion path connecting the plurality of wells sequentially between the supply unit and the drain. The method of claim 13, wherein the cell culture plate includes a plurality of supply parts and a plurality of drains, and the cell culture plate has an upper flow path connecting between two neighboring wells and inlets of two neighboring wells and two neighboring wells. A bioreactor comprising a lower flow path connecting outlets of two neighboring wells therebetween, wherein each of the plurality of supply units is connected to the upper flow path, and each of the plurality of drains is connected to the lower flow path. [Claim 17] The bioreactor according to claim 16, wherein electrodes for applying electrical stimulation are positioned in each of the wells. According to claim 17,
The bioreactor further comprises a cage for holding the electrode in the well. According to claim 13,
The bioreactor, characterized in that the inlet or the outlet is covered with a stopper structure in the form of a screen, a semi-permeable membrane, a woven or non-woven material, a micro/nano fiber sheet, or a gel. A cell culture plate comprising a plurality of wells; and
one or more recirculation systems coupled with the cell culture plate for recirculation or replacement of medium in the wells;
The cell culture plate.
a plurality of wells;
A supply unit for supplying recirculated or fresh medium from the outside;
An inlet formed on the inner upper portion of each well and into which a medium is injected;
An outlet formed on the inner lower part of each well;
A cell culture plate assembly comprising a drain through which medium to be recycled is discharged to the outside. According to claim 20,
The medium circulation system,
a badge reservoir;
A supply line connected to the supply unit;
A discharge line connected to the drain;
and a circulation pump supplying the medium in the medium reservoir to the cell culture plate through the supply line and circulating the medium to recover the medium in the cell culture plate to the medium reservoir through the discharge line. plate assembly. According to claim 20,
The cell culture plate assembly, characterized in that it comprises a perfusion path connecting the plurality of wells sequentially between the supply unit and the drain. According to claim 20,
The cell culture plate includes a plurality of supply parts and a plurality of drains, and the cell culture plate includes an upper flow path connecting inlets of two neighboring wells between two neighboring wells and two neighboring wells between two neighboring wells. A cell culture plate assembly comprising a lower flow path connecting between outlets of the plurality of supply units, each of which is connected to the upper flow path, and wherein each of the plurality of drains is connected to the lower flow path.

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