KR20120139399A - Utrasonic stimulated perfusion flow bioreactor system for culturing adult stem cell - Google Patents

Abstract

PURPOSE: A novel transonic stimulated perfusion flow culture chamber system for culturing adult stem cells in vitro to be similar to in vivo is provided to use a three-dimensional culture without applied stress and to optimize cell proliferation and differentiation. CONSTITUTION: A perfusion flow culture system comprises: a fresh medium container(8) for feeding fresh media; a control-based vessel(6), which includes a stirrer and a pH, DO measuring unit and controls the fresh media before circulating the media; a culture chamber(3) which provides a space for culturing cells and directly cultures the cells; a sensor(4) for measuring a cell environment; a 4-gas mixture control unit(1)(2) for CO_2, O_2, and N_2 which maintains the pH and DO of the media and optimizes a cell culture environment; and a waste vessel(9) which selectively discharges used media. [Reference numerals] (1) Gas mass flowmeter; (10) Cell stimulator; (11) Load cell transmitter; (2) Gas exchanger; (3) Culture chamber; (4,7) Sensor; (5) Inverted microscope; (6) Control-based vessel; (8) Fresh medium container; (9) Waste medium container; (AA) Medium line; (BB) Gas line

Description

Ultrasonic Stimulation Perfusion Culture System for Adult Stem Cells {UTRASONIC STIMULATED PERFUSION FLOW BIOREACTOR SYSTEM FOR CULTURING ADULT STEM CELL}

The present invention relates to a biomimetic perfusion culture chamber system for promoting in vitro differentiation of adult stem cells using ultrasonic stimulation as a biomechanical stimulation technique.

Conventionally, the study of the effect of mechanical stimulation on the shear stress applied to the fluid flow to the cell has recently been important as part of the study to biomimetic the in vivo culture environment. In vivo mechanotransduction is part of the energy conversion that mechanical stimulation of a cell converts it into a biochemical signal. Mechanical stimulus effects, such as shear stress applied to cells, have been reported to be some of the most important stimuli by enhancing mechanical transfer activity. In addition, in the research of the medical field to differentiate stem cells, which are the source of living cells, into many different kinds of living cells, biochemical treatment through the composition of culture medium and treatment of differentiation inducing substances to promote differentiation. In addition, various mechanical treatments such as tensile force, shear force, compression force, and ultrasonic waves are applied to cells.

Cells contained in mechano-responsive tissues of humans and animals are exposed to various mechanical stimuli such as tensile, shear, and compressive forces, and in articular cartilage tissues, repetitive mechanical stimuli are associated with arthritis ( causes diseases such as arthritis. Therefore, recent studies on cellular responses induced by mechanical stimulation have attracted attention.

In addition, in order to differentiate stem cells from stem cells into replacement tissues for humans and animals, especially machine-responsive tissues, to fully functional tissues, mechanical stimulation is applied to the cells in culture during stem cell culture. It is necessary to authorize. In this case, the degree of stimulation applied to the cells varies depending on the type of cells and the type of tissue to be differentiated.

In order to study cellular responses and stem cell differentiation, studies on mechanical stimuli such as tensile force, shear force, and compressive force have been performed. For example, Korean Patent Laid-Open Publication No. 10-2006-0134264 describes a biochip for cell stimulation and detection that can use ultrasonic waves as a mechanical stimulus for inducing differentiation of stem cells. In addition, Korean Patent Laid-Open No. 10-2004-0028700 discloses an apparatus for applying different localization to a cell by using a magnetic force to promote differentiation of a specific tissue from stem cells. In addition, Korean Patent Laid-Open No. 10-2002-0015575 describes a tissue regeneration using an abstract concept of mechanical stimulation. In addition, Korean Patent Laid-Open Publication No. 10-2008-0004881 discloses bone marrow-derived mesenchymal stem cells, characterized in that the bone marrow-derived mesenchymal stem cells are sonicated to improve cell adhesion and proliferation of mesenchymal stem cells. A method of obtaining is disclosed, and a method of treating at a rate of 5-50 minutes / day at the beginning of culture for 1-12 days, a method of treating at an intensity of 10-1000 mW / cm 2 by ultrasonic wave, and the like are disclosed.

However, the stimulation method cannot apply mechanical stimulation to cells cultured in the apparatus, and can only target a small number of cells, thereby failing to obtain sufficient cells for analysis of the cells.

On the other hand, in relation to biotissue engineering, many efforts have been made to culture animals' cells and tissues in vitro. However, when culturing the cells and tissues of animals in vitro, the culture efficiency is lower than in vivo culture, and also depends on expensive nutritional medium in the culture. In particular, culturing adult stem cells is more difficult than culturing cells and tissues of general animals, and the development of a system capable of three-dimensionally culturing these adult stem cells in vitro is required. In this regard, the perfusion bioreactor system for culturing adult stem cells can be applied for proliferation and differentiation of cells to regenerate three-dimensional tissues in vitro, and is particularly important as a support device for organ culture of organs. It is becoming. In general, the cell culture system is designed to provide an in vitro environment in which cells can grow. The culture system in a perfusion flow type has a general environment, that is, a temperature of 37 ° C. and CO _2. Although it is done in 5% environment, it is not possible to control environment of precise cell growth (precise measurement sensor and gas control) and image monitoring function of real time cell culture.

Korean Patent Registration No. 849516 discloses a cell culture chamber and a cell culture apparatus including the same, wherein the cells to be cultured can be cultured in a uniformly distributed state without being influenced by the inflow and outflow of the culture solution into the cell culture chamber. Disclosed is a configuration capable of controlling the amount of supply of gas and culture solution required for cell culture to a culture chamber. However, no specific control method has been described, and no biomechanical stimulation technique for promoting cell culture is used, making it difficult to obtain cell culture as much as desired.

The present inventors have conducted extensive research in view of the above, as a result of perfusion culture that can be controlled to enable the operation of a comprehensive cell culture environment, such as oxygen transfer, pH, DO, temperature, mechanical stimulation as well as sterilization consideration The present invention has been completed by developing a chamber system, using ultrasonic stimulation as a biomechanical stimulation method, and finding an optimal cell proliferation method according to its intensity and stimulation time.

Accordingly, an object of the present invention is to provide a biomimetic ex vivo cell culture system by developing a new perfusion culture chamber system for in vitro culture of adult stem cells similar to in vivo. In addition, the present invention uses an ultrasonic stimulation as a biomechanical stimulation method for promoting the differentiation of adult stem cells, and to examine the proliferation of cells according to the intensity and stimulation time, to provide an optimal ultrasonic stimulation method do. In addition, an object of the present invention is to provide a new perfusion culture chamber system by optimizing the proliferation and differentiation of cells by applying the optimal ultrasonic stimulation method to a new perfusion culture chamber system.

In the present invention, the perfusion culture chamber system includes a 4-gas mixture controller of CO ₂ , O ₂ , N ₂ , air, a control medium container and a culture chamber, wherein the control medium container and the culture chamber are the 4-gas. The 4-gas mixture is supplied from the mixture control unit, the culture chamber is supplied with the culture medium in which the culture environment is controlled from the control medium container, and the cell culture of the culture chamber is continuously monitored.

In one embodiment, the control medium container can receive the medium from the fresh medium container, the medium discharged from the culture chamber can be recovered and reused in the control medium container.

In one embodiment, the culture chamber may comprise a reversed phase microscope and may further comprise a cell stimulator.

In one embodiment, the cell stimulator included in the culture chamber may apply ultrasonic stimulation.

In one embodiment, the control medium container may include a pH sensor, DO sensor, ammonia sensor or temperature sensor.

In one embodiment, the culture chamber may include a pH sensor, DO sensor, CO ₂ sensor, O ₂ sensor, temperature sensor or humidity sensor.

In one embodiment, the cells cultured in the culture chamber may be adult stem cells.

In one embodiment, the culture chamber can be designed to have a constant working volume.

In one embodiment, the ultrasonic stimulation can have an intensity of 200 mW / cm ² or less, a duty cycle of at least 5% and less than 50%, or a stimulation time in the range of 1 minute / day to 10 minutes / day.

In one embodiment, the ultrasonic stimulation can have an intensity of 200 mW / cm ² or less, a duty cycle of 30% or less, and a stimulation time of 10 minutes / day.

In one embodiment the ultrasonic stimulation can have an intensity of 50 mW / cm ² , a duty cycle of 5%, and a stimulation time of 10 minutes / day.

In the present invention, by providing a new perfusion culture chamber system for in vitro culture similar to in vivo, it is possible to culture adult stem cells that are not easy to culture, furthermore, a new perfusion culture The optimal ultrasonic stimulation method is applied to the chamber system to provide a new perfusion culture chamber system with optimized cell proliferation and differentiation. In addition, the present invention provides a culture system capable of culturing the cells and tissues of animals in three dimensions in vitro without stress, can be applied to the production of artificial tissues using cell culture technology, biostimulation technology and the like. In addition, the present invention can be applied to the technology to supply nutrition and gas to the organ (organ), unlike the existing cell culture process, it can provide a three-dimensional culture system of the environment similar to the living human body.

1 is a diagram of a perfusion culture chamber system according to one exemplary embodiment.
2 is a schematic diagram of a culture chamber according to one exemplary embodiment.
3 is a diagram of a three-dimensional cell culture chamber according to one exemplary embodiment.
4 is a simplified view of the duty cycle.
5 is a diagram of a perfusion culture chamber system used in Examples 14-17 in accordance with one exemplary embodiment.
FIG. 6 is a diagram illustrating cell differentiation and growth of alveolar bone stem cells cultured according to Reference Examples 1 and 14 with an electron microscope. FIG.
7 is a view showing the number of cells cultured according to Example 1.
8 is a view showing the number of cells cultured according to Example 2.
9 is a view showing the number of cells cultured in accordance with Example 3.
10 is a view showing the number of cells cultured in accordance with Example 4.
11 is a view showing the number of cells cultured according to Example 5.
12 is a view showing the number of cells cultured according to Example 6.
13 is a view showing the number of cells cultured in accordance with Example 7.
14 is a view showing the number of cells cultured according to Example 8.
15 is a view showing the number of cells cultured according to Example 9.
16 is a view showing the number of cells cultured according to Example 10.
17 is a view showing the number of cells cultured according to Example 11.
18 is a view showing the number of cells cultured according to Example 12.
19 is a view showing the number of cells cultured according to Example 13.
20 is an electron microscope view of cell differentiation and growth on a three-dimensional scaffold of alveolar stem cells cultured according to Example 14;
Figure 21 shows the viability of the cells cultured in accordance with Examples 15 and 16.
22 is a diagram showing the viability of the cells cultured in Example 17.

EMBODIMENT OF THE INVENTION Hereinafter, embodiment of this invention is described in detail, referring drawings. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized, and other changes may be made, without departing from the spirit or scope of the subject matter presented herein. As described herein throughout and illustrated in the figures, the disclosure herein may be arranged, substituted, combined, and designed in a very wide variety of different configurations, all of which are expressly contemplated and form part of the present disclosure. The point will be easily understood.

In relation to biotissue engineering, a perfusion bioreactor system for culturing adult stem cells can be applied for the proliferation and differentiation of cells to regenerate three-dimensional tissues in vitro, and in particular, a support device for long-term culture of organs. Can be used as. Such a culture system needs to be controlled to enable the operation of a comprehensive proper cell culture environment such as oxygen transfer, pH, DO, temperature, mechanical stimulation as well as sterilization consideration, and an automatic operation step is required to enable this. The most important design factors when designing such a system are the consideration of cell adhesion, circulation of media, nutrient status, pH, temperature, dissolved oxygen (DO) in culture medium, CO ₂ , O ₂ , fluid Consideration should be given to the rate of perfusion and stimulation techniques such as mechanical stimulation.

Figure 1 shows a simplified process diagram of the perfusion culture system of the present invention briefly. The perfusion culture system of the present invention is a fresh medium container (8) for supplying fresh medium, a control medium container for controlling the fresh medium before circulating the medium (including the control based vessel, agitation and pH, DO, etc. measurement unit) (6 ), A culture chamber 3 for culturing the cells directly, a sensor 4 for measuring the cell environment, a CO for maintaining the pH and DO of the medium and optimizing the cell culture environment. ₂ , O ₂ , N ₂ , 4-gas mixture control of air ((1) and (2)), waste vessels (9) to select and discharge the medium used according to the appropriate conditions, etc. Can be configured.

In FIG. 1, the 4-gas mixture controller is used to control the culture environment of cells in the culture chamber system, and is used to control four gases such as air, N ₂ , CO ₂ , and O ₂ . May be used. Specifically, in the present invention, the four-gas mixture controller may include a four gas mass flow controller 1 and a gas exchange device 2. 4 Gas mass flow meters (1) include air, N ₂ , CO ₂ and O ₂ The source of is connected, and the flow rate of air, N ₂ , CO ₂ and O ₂ can be adjusted at a rate of 0 to 500CC / min. In addition, a gas exchange device 2 is provided between the four gas mass flow meter 1 and the culture chamber 3 to provide air, N ₂ , CO ₂ , and O ₂ in the culture chamber 3. It is possible to adjust gas components, such as these suitably. In addition, the air discharged from the gas exchange device (2), N ₂ , CO ₂ , O ₂ The gaseous components such as these are supplied not only to the culture chamber 3 but also to the control medium container 6, so that the medium environment in the control medium container 6 can be appropriately adjusted to an optimum state.

In FIG. 1, the culture chamber 3 is a space in which cells are directly cultured, and gas is supplied from a 4 gas mixture control unit, and fresh medium is supplied from a control medium container 6 to maintain an optimal cell culture environment. . In addition, the control medium container 6 and the culture chamber 3 may be equipped with a precision peristaltic pump for perfusion flow rate adjustment, and after the fresh medium is introduced into the culture chamber 3, the used medium is again controlled medium container. (6) can be recovered. A precision peristaltic pump may be installed between the culture chamber 3 and the control medium vessel 6 for discharge and / or recovery of the used medium, and the precision peristaltic pump may be connected to a control unit in a desired range. It can be adjusted automatically within. In addition, the used medium may be discharged directly from the culture chamber 3 to the waste medium container 9, and a precision peristaltic pump for adjusting the perfusion flow rate may be installed between the culture chamber 3 and the waste medium container 9. The precision peristaltic pump may be connected to a control unit and automatically adjusted within a desired range.

In addition, the culture chamber 3 may be provided with a sensor 4 to detect pH, DO, CO ₂ , O ₂ and temperature, humidity and the like to monitor the culture environment of the cell in real time. An image display unit (not shown) that can display information measured from 4) can be provided. In addition, a pressure transmitter for measuring the internal pressure of the culture chamber 3 may be installed, and the numerical values of the sensor 4 and the pressure transmitter may be adjusted to a desired numerical range through the control unit.

In the present invention, the upper lid portion and the lower bottom portion of the culture chamber 3 may be made of glass, and it is particularly preferable that a spherical glass serving as a window for monitoring the cell culture is installed at the bottom. A reversed phase microscope (5) can be installed at the bottom of the bottom to continuously monitor the cell culture in real time, and the cell image of the stem cells to be cultured can be configured using a phase contrast microscope (e.g., Nikon TS100, Japan) and a digital camera. (E.g., SocpeTek DCM200, China). Perfusion culture chamber system of the present invention, by introducing the real-time monitoring system, it can provide an advantage that can be confirmed with the naked eye and the image of the culture of the cell.

In the perfusion culture chamber system of the present invention, a biomechanical stimulation method for promoting the differentiation of stem cells may be used, and the cell stimulator 10 is installed at the lower end of the culture chamber 3 to generate fluid from the perfusion flow. In addition to the shear force, stimulation methods such as ultrasonic waves, microcurrents, electromagnetic fields, and the like may be further used.

The perfusion culture chamber system of the present invention includes a control based vessel 6 for controlling and maintaining the medium environment supplied to the culture chamber 3. The control medium container 6 is connected to an input for medium perfusion of the culture chamber 3, and is configured to control the fresh medium before circulating the medium. In addition, the control medium container is provided with a sensor capable of measuring pH, DO, ammonia, temperature, etc., a stirrer may be installed at the top for uniformity of the fresh medium in the control medium container (6), the stirrer A stirring motor may be installed.

The control medium container 6 is connected to the medium input of the culture chamber 3, and can continuously supply fresh medium to the culture chamber through a variable metering pump. As an embodiment, a variable speed peristaltic pump may be used as the variable metering pump.

The fresh medium supplied to the culture chamber 3 is supplied using the metering pump capable of precise control, and may be supplied at a perfusion amount of about 0.03 to 0.3 ml / min. The perfusion amount may vary depending on the cells to be cultured, and is not limited as long as it can give a favorable shear stress to the cells and provide a positive environmental effect on growth.

In addition, in the present invention, the control medium container 6 can be connected through the medium output part of the culture chamber 3. That is, in the present invention, the control medium container 6 can be connected to both the medium input part and the output part, and the unused medium discharged from the medium output part of the culture chamber 3 is supplied to the control medium container 6, so that it is not used. Reuse of the medium may be possible. Unlike the medium for culturing microorganisms or cells, the medium used for culturing stem cells is quite expensive because various nutrients such as NaCl, NaHCO ₃ , D-glucose and L-glutamine must be included. In addition, since the nutrient components of the medium of the medium outlet portion of the culture chamber 3 are often discharged in an unused state, the perfusion type culture chamber system of the present invention also secures economical efficiency by recycling the unused medium of the outlet portion. can do. Further, in the present invention, a sensor 7 capable of measuring ammonia, DO, pH, etc. may be provided in the control medium container, and when the by-products of the medium recovered from the outlet part increase or the consumption of nutrient components is large, It can be controlled to be discharged from the control medium container 6 to the waste medium container 9 through the control unit, and can also be adjusted to receive oxygen or the like from the four gas mixture control unit. The discharge of the medium from the control medium container 6 to the waste medium container 9 can be controlled via an optionally equipped pump system. Specifically, when the pH is 5 or less, or the oxygen content at the time of DO measurement is 10 ppm or less, it is preferable that a part or more of the medium is discharged from the control medium container 6 to the waste medium container 9. At this time, the fresh medium can be supplied by receiving the signal of the load cell transmitter as much as the discharged medium amount.

In addition, in the present invention, the control medium container 6 and the unused fresh medium container 8 can be connected to appropriately control the medium environment in the control medium container 6. At the bottom of the control medium container 6, a load cell transmitter 11 capable of measuring the total weight of the control medium container may be installed, and the amount of medium discharged to the waste medium container 9 may be adjusted. Thus, an appropriate amount of unused fresh medium from the fresh medium container 8 can be replenished to the control medium container 6.

Hereinafter, look at in more detail with reference to Figure 2 for the structure of the culture chamber used in the present invention. Figure 2 briefly shows the design of the culture chamber used in the present invention. As described above, the culture chamber used in the present invention is designed to monitor the culture environment of the cell in real time, and the sensors such as pH, DO, CO ₂ , O ₂ and temperature, humidity to enable the cell environment control Install and collect data in real time for analysis. In addition, since the culture chamber is injected with fresh medium and used for cell culture, it must be continuously exchanged to keep the cell tissue fresh. Therefore, the culture chamber and the culture chamber system including the same have a continuous supply and discharge process. It is essential that the configuration can be performed smoothly, in the present invention can be precisely adjusted and maintained by the above-described metering pump.

In particular, the culture chamber used in the present invention may be mechanically designed to precisely maintain a working volume at about 3 ml even when the medium is continuously supplied to the culture chamber through a variable precision peristaltic pump. .

In addition, the culture chamber used in the present invention may be designed in a water-cooled structure for maintaining a precise temperature, for this purpose may include an inlet and outlet of water. Preferably, the culture chamber used in the present invention has a structure capable of mounting the three-dimensional cell culture chamber of FIG. 3 inside the cell culture chamber of FIG. 2, and has a structure capable of precisely controlling the three-dimensional cell culture. In addition, the culture chamber used in the present invention may include the inlet and outlet of the mixed gas, the installation portion of the various sensors, in addition to the inlet and outlet for the perfusion of the medium.

In addition, the culture chamber used in the present invention is one of the most important design factors, in consideration of autoclaving, as shown in Figure 2, the lid portion, the chamber portion to facilitate the retrieval (reconstruction) of various sensors of the culture chamber system as shown in Figure 2 It is desirable to have a structure in which the bottom part, the suture part, the entrance and exit part, etc. can be easily separated.

In addition, in the perfusion culture chamber system of the present invention, a biomechanical stimulation method for promoting the differentiation of stem cells can be used. Various cells constituting the tissues in the human body are subject to a certain physical effect, vascular endothelial cells due to the flow of blood is mainly affected by shear stress, compressive load in cartilage cells, tensile stimulation in ligament cells. In order to provide an environment similar to a real living body in vitro and to improve the ability to adapt to a living body, a system for directly adding a physical stimulus using a bioreactor to a cell was introduced (Sodian et al., 2002). The physical environment outside the living body has a lot of effects on the function of the proliferation and differentiation of cells, and many studies have been made. As such, each tissue in vivo is constantly receiving specific physical stimuli, and the specific physical stimulus may also affect the differentiation and formation of tissues. Therefore, in the perfusion culture chamber system, physical and mechanical stimuli to promote cell growth and differentiation can be used along with general chemical stimuli.

In the present invention, various mechanical treatment methods such as tensile force, shear force, compression force, and ultrasonic wave, as well as biomechanical stimulation methods such as microcurrent, electromagnetic field, and magnetic field stimulation can be used in various ways, and a plurality of combinations of the stimulation methods are possible.

In particular, the ultrasonic stimulation method of the biomechanical stimulation method can be used in the present invention. The ultrasonic stimulation system used in the present invention may be any conventionally known ultrasonic stimulation system, and is not particularly limited.

Typically, the ultrasonic stimulation system is composed of a main body, an ultrasonic stimulation unit and a power supply unit. Also, in general, ultrasonic waveforms are divided into continuous mode and pulse mode, and duty cycle is defined in pulse mode, which is the time per cycle when the square wave is at the high level. The pulse duty cycle is defined as follows.

Duty cycle = {(pulse width) / (cycle)} × 100

4 is a simplified illustration of the duty cycle for the purpose of understanding the equation. Here, the pulse width means the time from the threshold value 50% of the pulse rising section to the threshold value 50% of the next falling section.

The present inventors have studied the ultrasonic stimulation as a biological stimulus, only the ultrasonic stimulation of the appropriate intensity can have a positive effect on the proliferation of the cell, and may adversely affect the proliferation of the cell if not appropriate conditions It was confirmed. In a culture plate used for cell culture, when ultrasonic waves are stimulated at various intensities, the cells do not grow well in the center portion of the plate culture, and the cells grow better in the edge portion. In other words, the cells grow better from the center to the edge. In addition, the cells at the edges of the nucleus are distinct and each cell grows larger and thicker than the control group without ultrasonic stimulation. This is expected to cause the cells to move to the edge due to ultrasonic stimulation in the central portion of the plate culture, which is just above the ultrasonic transducer. Therefore, only ultrasonic stimulation of appropriate intensity may have a positive effect on cell proliferation, and it is not easy to find an ultrasound condition optimized for cell differentiation and proliferation.

As a result of the study by the present inventors, the ultrasonic stimulation used in the present invention is preferably low intensity, the ultrasonic stimulation of a suitable distance from the ultrasonic sensor is preferable for cell proliferation, and if the ultrasonic intensity is too strong, it may rather adversely affect the cell culture. have. Specifically, the ultrasonic intensity used in the present invention can be adjusted to 200 mW / cm ² or less, 300 mW / cm ² These stimuli are too strong and may adversely affect the proliferation of adult stem cells.

In addition, the ultrasonic stimulation used in the present invention is preferably used within a range having a specific duty cycle. Specifically, the ultrasonic waves used in the present invention may be adjusted to a duty cycle of 5% or more and less than 50%, and preferably to a duty cycle of 5% or more and 30% or less. At 50% or more of the duty cycle, they may adversely affect the proliferation of stem cells.

In addition, the ultrasonic stimulation used in the present invention, the conditions of the stimulation time 1 minute / day to 10 minutes / day is preferable. Longer stimulation times such as 20 minutes / day and 30 minutes / day may adversely affect cell proliferation.

Therefore, when proper combination of duty cycle of 30% or less and low intensity ultrasound intensity of 200 mW / cm ² or less at 10 minutes / day of ultrasonic stimulation time is appropriate, it is preferable for proliferation of adult stem cells, and 50 mW / cm ² intensity, 5 Low intensity ultrasound stimulation at% duty cycle, 10 min / day stimulation conditions is more preferred for proliferation of stem cells.

On the contrary, even if the same ultrasonic stimulation intensity is different depending on the duty cycle and the stimulation time, the effect on the proliferation of adult stem cells, the higher the ultrasonic intensity is more preferably a combination of low duty cycle and stimulation time.

The present invention provides a novel perfusion culture chamber system for culturing adult stem cells in vitro similar to in vivo, and furthermore, applies an optimal ultrasonic stimulation method to the new perfusion culture chamber system. This provides a new perfusion culture chamber system with optimized cell proliferation and differentiation. In addition, the present invention provides a culture system capable of culturing the cells and tissues of animals in three dimensions in vitro without stress, can be applied to the production of artificial tissues using cell culture technology, biostimulation technology and the like.

The following examples are intended to further illustrate the features and advantages of the subject matter of the present invention, but are not limited to the following examples. The subject matter of the present invention should not be considered limited to the specific embodiments and examples described herein. In view of the present disclosure, in addition to various exemplary embodiments and embodiments, those skilled in the art will readily recognize that modifications, substitutions, additions, and combinations of some of the configurations disclosed herein are possible.

[ Example ]

Reference Example  One

Alpha-MEM medium to 10% SERUM (FBS, Welgene Inc., Korea ) and 37 ℃ using the culture medium into the ascorbic acid (L-ascorbic acid), antibiotics, and sodium bicarbonate of 10 nM, 5% CO ₂ concentration, and Adult stem cells were statically cultured in an incubator with a relative humidity of 95% (Steri-Cycle 370 Incubator, Thermo Fisher Scientific. USA). Stem cells were the alveolar bone marrow-derived stem cells collected by Seoul National University College of Dental Biointerface Engineering Laboratory. After incubation for about 4 days, the differentiation and growth of the cells were observed, and the results of 4 replicates are shown in FIG. 6 (upper control).

In addition, the survival of alveolar bone cells was analyzed using the WST-1 assay (EZ-Cytox Cell Viability Assay Kit, Daeillab Service Co., LTD). The assay is a staining method in which tetrazolium salts are added to a culture medium to analyze living cells. The principle is that tetrazolium salt WST-1 utilizes a property of forming water-soluble formazan by a mitochondrial reductase in a cell. The amount of marzan is directly related to the number of living cells. The amount of water-soluble formazan was quantified by measuring the absorbance at 460 nm with a multiple spectrophotometer (Victor 3, Perkin Elmer, USA).

Survival of alveolar bone cells is shown in FIG. 21 (see static).

Cell Proliferation Effect of Ultrasound Stimulation Intensity

Example  One

The following experiment was conducted to confirm the effect of the ultrasonic stimulation on the culture of stem cells as a biostimulation.

Stem cells derived from alveolar bone marrow collected by Seoul National University College of Dentistry and Biointerface Engineering Laboratory at 37 ° C., 95% moisture, and 5% CO ₂ were incubated in an incubator and 3.0 × 10 ⁴ cells / in a 100 mm culture plate. Seeding in ml and incubated for 24 hours was used for ultrasonic stimulation.

Ultrasound was irradiated at an intensity of 50, 100, 200, 300, 400, 500 mW / cm ² at a frequency of 1 MHz, 50% duty cycle, 10 minutes / day of stimulation time was kept the same.

In order to prevent contamination of the cells, the ultrasonic stimulator in the incubator was sterilized with 70% ethanol, and the ultrasonic stimulator was placed in the incubator for 3 days under conditions of 37 ° C, 95% relative humidity, and 5% CO ₂ . Ultrasound stimulation was applied. The control group was not incubated with the ultrasonic stimulus (that is, the ultrasonic intensity 0) was also incubated for 3 days in the same state.

An ultrasonic gel (Choongwae Pharma Co., Korea) was used to provide effective ultrasonic stimulation without contacting air between the bottom of the 100 mm petri dish and the ultrasonic transducer.

After completion of the culture, cells were harvested using trypsin-EDTA (Gibco, USA) which is generally used for cell number measurement. In order to check the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in Figure 7, when the ultrasound was stimulated at 50, 100, 200 mW / cm ² intensity alveolar bone marrow-derived stem cells proliferated well compared to the control, 300 mW / cm ² intensity from the control to the control Cell number decreased. Especially for 400 mW / cm ² and 500 mW / cm ² intensity, the number of cells decreased significantly.

Thus, at 50% duty cycle, ultrasonic stimulation of 200 mW / cm ² or less intensities promotes proliferation of alveolar bone marrow-derived stem cells, while ultrasonic stimulation of 300 mW / cm ² or more is strong and is rather effective in stem cell proliferation. It was confirmed that the adverse effect.

ultrasonic wave Duty  Cell proliferation effect of the cycle

Example  2

To determine the effect of ultrasonic stimulation of various duty cycles on the proliferation of alveolar bone marrow-derived stem cells, the degree of cell proliferation by duty cycle was measured at 10 minutes / day of stimulation.

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200 mW / cm ² at a frequency of 1 MHz, the duty cycle 5%, stimulation time 10 minutes / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in FIG. 8, 100 mW / cm ² Stimulation of alveolar bone marrow-derived stem cells proliferated with a slight difference compared to the control group when stimulated by intensity, and the number of cells increased by about 136% and 157%, respectively, at 50 mW / cm ² and 200 mW / cm ² intensity stimulation. It was. For 300 mW / cm ² intensity, the number of cells was rather reduced.

Through this, ultrasonic stimulation of less than 200 mW / cm ² intensity at 5% of the ultrasonic duty cycle was able to confirm the proliferation effect of the alveolar bone marrow-derived stem cells at a significant level of 5% compared to the control. However, ultrasonic stimulation of 300 mW / cm ² intensity was found to be adversely affecting the proliferation of alveolar bone marrow-derived stem cells.

Example  3

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, the duty cycle 10%, stimulation time 10 minutes / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in Figure 9, the number of cells increased by about 138% compared to the control at 50 mW / cm ² intensity stimulation, alveolar bone marrow-derived stem cells compared to the control even when stimulated at 100 mW / cm ² intensity Proliferation of was promoted. Stimulation at 200 mW / cm ² intensity decreased the cell number compared to the control group, and significantly decreased for 300 mW / cm ² intensity.

Through this, it was confirmed that the ultrasonic stimulation of less than 100 mW / cm ^{2 in the} duty cycle 10% promotes the proliferation of alveolar bone marrow-derived stem cells, the ultrasonic stimulation of 300 mW / cm ² intensity is strong and rather alveolar bone It was confirmed to adversely affect the proliferation of bone marrow-derived stem cells.

Example  4

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, the duty cycle 30%, stimulation time 10 minutes / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using a trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in Figure 10, when the stimulation at 100 mW / cm ² intensity alveolar bone marrow-derived stem cells proliferated about 132% compared to the control group, the number of cells decreased for 200, 300 mW / cm ² intensity.

Through this, it was confirmed that the ultrasonic stimulation of 100 mW / cm ² intensity in the 30% duty cycle promotes the proliferation of alveolar bone marrow-derived stem cells at a significance level of 5%, the ultrasonic stimulation of intensity 200 mW / cm ² or more It was confirmed that adversely affect the proliferation of alveolar stem cells.

Example  5

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, 50% duty cycle, 10 minutes / day stimulation time was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to check the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in Figure 11, when stimulated at 100, 200 mW / cm ² intensity, alveolar bone marrow-derived stem cells proliferated little compared to the control group, the number of cells decreased for 300 mW / cm ² intensity It was.

Through this, in case of 50% duty cycle, the use of ultrasonic stimulation with intensity of 200 mW / cm ² or less did not show a significant effect on the proliferation of alveolar bone marrow-derived stem cells, and the ultrasonic stimulation with intensity of 300 mW / cm ² or more was rather It has been confirmed to adversely affect the proliferation of alveolar bone marrow-derived stem cells.

Cell Proliferation Effect of Ultrasound Stimulation Time

Example  6

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, the duty cycle 10%, stimulation time 1 minute / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in Figure 12, when the stimulation at 100, 200 mW / cm ² intensity was little promoted proliferation of alveolar bone marrow-derived stem cells compared to the control, when stimulated at 300 mW / cm ² intensity Alveolar bone marrow-derived stem cells proliferated about 147% compared to the control group.

Thus, in the case of 1 minute stimulation time, the use of ultrasonic stimulation with intensity of 200 mW / cm ² or less did not show a positive effect on the proliferation of alveolar bone marrow-derived stem cells, and the ultrasonic stimulation of 300 mW / cm ² intensity was alveolar bone Although it has a positive effect on the proliferation of bone marrow-derived stem cells, it is thought that it is possible to increase the stimulation time at a weaker intensity.

Example  7

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, the duty cycle 10%, stimulation time 3 minutes / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in Figure 13, when the stimulation at 100, 200 mW / cm ² intensity, the proliferation of alveolar bone marrow-derived stem cells was hardly promoted compared to the control, when the stimulation at 300 mW / cm ² intensity Alveolar bone marrow-derived stem cells were decreased compared to the control group.

Through this, when the stimulation time of 3 minutes using ultrasonic stimulation of 200 mW / cm ² or less intensity does not show a positive effect on the proliferation of alveolar bone marrow-derived stem cells, ultrasonic stimulation of 300 mW / cm ² or more intensity is alveolar bone It was confirmed that the negative effect on the proliferation of bone marrow-derived stem cells.

Example  8

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, the duty cycle 10%, stimulation time 5 minutes / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in Figure 14, when stimulated at 50, 100, 200 mW / cm ² intensity, about 125%, about 119%, about 125% alveolar bone marrow-derived stem cells proliferated compared to the control, respectively, 300 mW At / cm ² intensity, cell number was decreased compared to the control.

Through this, 5 minutes of stimulation time was shown to have a positive effect on the proliferation of alveolar bone marrow-derived stem cells by using ultrasonic stimulation of 200 mW / cm ² or less, the ultrasonic stimulation of 300 mW / cm ² or more is derived from alveolar bone marrow It was confirmed that the negative effect on the proliferation of stem cells.

Example  9

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, the duty cycle 10%, stimulation time 10 minutes / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using a trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in Figure 15, about 138% alveolar bone marrow-derived stem cells proliferated compared to the control group when stimulated at 50 mW / cm ² intensity. In the case of stimulation at 100 mW / cm ² intensity, alveolar bone marrow-derived stem cells proliferated with a slight difference compared to the control, and the number of cells decreased at 200 mW / cm ² intensity stimulation. For 300 mW / cm ² intensity, a significant decrease in cell number was confirmed. Therefore, it was necessary to experiment under the condition that the duty cycle (10%) and the stimulation time (10 minutes) were further increased at 100 mW / cm ² intensity condition.

Example  10

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, the duty cycle 30%, stimulation time 10 minutes / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in Figure 16, when the stimulation at 100 mW / cm ² intensity alveolar bone marrow-derived stem cells proliferated about 132% compared to the control group, the number of cells from the 200 mW / cm ² intensity stimulation was reduced compared to the control group It was.

Through this, the ultrasonic stimulation of 100 mW / cm ² intensity at 30% duty cycle, 10 minutes of stimulation time shows the proliferation effect of the alveolar bone marrow-derived stem cells, the ultrasonic stimulation of 200 mW / cm ² or more intensity is alveolar bone marrow-derived stem It was confirmed that the negative effect on the proliferation of the cells.

Example  11

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, the duty cycle 30%, stimulation time 20 minutes / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in FIG. 17, when stimulated at 100 mW / cm ² intensity, alveolar bone marrow-derived stem cells proliferated about 113% compared to the control group, and the number of cells decreased from 200 mW / cm ² intensity stimulation. It was.

Comparing FIG. 16 and FIG. 17, the proliferative effect of alveolar bone marrow-derived stem cells was lowered at 100 mW / cm ² , duty cycle 30%, and stimulation time of 20 minutes compared to stimulation time of 10 minutes under the same conditions.

Example  12

Alveolar bone marrow-derived stem cells were cultured under the same conditions as in Example 1 except that the ultrasonic stimulation conditions were changed as follows. Ultrasound was irradiated at an intensity of 50, 100, 200, 300 mW / cm ² at a frequency of 1 MHz, the duty cycle 30%, stimulation time 30 minutes / day was kept the same. The control group was also cultured in the same state without the ultrasonic stimulation (ie, ultrasonic intensity 0).

In order to confirm the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in Figure 18.

As can be seen in Figure 18, the number of cells from the stimulation of 50 mW / cm ² intensity was reduced compared to the control, the number of stimulation of 100 and 200 mW / cm ² intensity also decreased compared to the control, 300 mW / cm ² In strength, the number of cells decreased significantly.

Through this, it was confirmed that applying too long ultrasonic stimulation of 30% duty cycle and 30 minutes of stimulation time had a negative effect on proliferation of alveolar bone marrow-derived stem cells.

Therefore, the ultrasonic stimulation experiments showed the proliferation effect of the alveolar bone marrow-derived stem cells at 50 mW / cm ² intensity at a duty cycle of 10% and a stimulation time of 10 minutes.

Example  13

Pulp stem cells were used instead of alveolar bone marrow-derived stem cells as adult stem cells, and pulp stem cells were cultured under the same conditions as in Example 1. However, the ultrasonic stimulation conditions at 10 minutes stimulation time, 50 mW / cm ² intensity, 5%, 10%, 30%, 50% duty cycle was confirmed the degree of proliferation of cells during the ultrasonic stimulation experiment.

In order to confirm the number of living cells using trypan blue staining method, using a hemocytometer counting the number of cells by measuring the cell proliferation, the results are shown in FIG.

As can be seen in FIG. 19, the cell number was increased by about 150% compared to the control at 5% duty cycle stimulation, the cell number was increased by about 134% at 10% duty cycle, and at 30% duty cycle. The number of cells increased about 114% at 131% and 50% duty cycle stimuli.

Through this, it was confirmed that the intensity of 50 mW / cm ² , 10 minutes stimulation time has a positive effect on the proliferation of alveolar bone marrow-derived stem cells in all duty cycles. In addition, a 50 mW / cm ² intensity, 5% duty cycle low intensity ultrasound stimulus was given for 10 minutes every 3 days, resulting in the proliferation of pulp stem cells up to about 150%. Ultrasound stimulation was found to be effective for cell proliferation. This is similar to the result that alveolar bone marrow-derived stem cells were able to proliferate alveolar bone marrow-derived stem cells up to about 136% after 10 minutes of 50 mW / cm ² intensity and 5% duty cycle low intensity ultrasound stimulation for 3 days. to be.

Accordingly, it was confirmed that low-intensity ultrasound stimulation at 50 mW / cm ² intensity, 5% duty cycle, and 10 min / day stimulation conditions is a very effective method for proliferation of other stem cells and animal cells.

Example  14

To confirm the applicability of low-intensity ultrasound stimulation in 3D scaffold cultures, low-intensity ultrasound stimulation was performed under conditions of 50 mW / cm ² , 10% duty cycle, and 10 min stimulation time effective for alveolar bone marrow-derived stem cells. After the treatment and nanofiber culture, the growth of alveolar bone marrow-derived stem cells was observed with an electron microscope, and is shown in FIG. 20.

Through this, it was confirmed that the application of low-intensity ultrasound stimulation in the culture of the nano support of stem cells.

Perfusion Culture Chamber System

Example  15

A perfusion culture system for culturing human alveolar bone marrow-derived stem cells, one of the adult stem cells, was designed as shown in FIG. 5.

Specifications of the variable metering pump used in the perfusion culture system are shown in Table 1 below.

Item Specifications Silicone tubing 1.6 mm wall tubing Pressure 2.4 bar with silicone tubing Flow rate (ml / revolution) 0.001 (ID: 0.13 mm) Flow rate 34 ml / min Flow rate 0.01 ml / min

In the perfusion culture system, 500 ml of control culture vessel, 500 ml of fresh medium vessel, and 1000 ml of waste medium vessel were used, and the medium was Alpha-MEM (LM 008-01, Welgene Inc. manufactured by Weljin Co., Ltd.). , South Korea) Culture medium containing 10% SERUM (FBS, Welgene Inc., Korea) and 10 nM of ascorbic acid (L-ascorbic acid), antibiotics and sodium bicarbonate was used.

Stem cells were the alveolar bone marrow-derived stem cells collected by Seoul National University College of Dental Biointerface Engineering Laboratory.

In the cell culture chamber, the temperature is around 37 ℃, the CO ₂ concentration is around 5%, the O ₂ concentration is 18-25%, the humidity is 80-95% RH, and the chamber pressure is 55 ~ The range was adjusted to 70 mBar.

In a controlled culture vessel, the temperature was controlled in the range of 37 ± 1 ° C, pH in the range of 7-8, DO in the range of 2-4%, and RPM of the stirring motor in the range of 100-400.

Medium flow rate in the perfusion culture experiment was maintained at about 0.3 ml / min using a variable metering pump.

After culturing the alveolar bone marrow-derived stem cells under the above conditions for about 4 days, the differentiation and growth of the cells were observed, and the results of 4 replicates are shown in FIG.

As shown in Figure 6, it was observed that the growth morphology ( cell morphology) of the cells in the culture of stem cells in the commercialized CO ₂ incubator (Reference Example 1) and the cell culture chamber system of the present invention is almost similar.

In addition, as in Reference Example 1, the survival of the alveolar bone cells was analyzed using the WST-1 assay (EZ-Cytox Cell Viability Assay Kit, Daeillab Service Co., LTD.). (See 0.3 ml / min). In the perfusion culture chamber system according to the present invention, when the medium flow rate was maintained at about 0.3 ml / min, it was possible to obtain the cell viability equivalent to the static culture.

Example  16

Alveolar bone marrow-derived stem cells were cultured for about 4 days under the same conditions as in Example 14 except that the medium flow rate was maintained at 0.03 ml / min, and then the differentiation and growth of the cells were observed.

In addition, as in Reference Example 1, the survival of the alveolar bone cells was analyzed using the WST-1 assay (EZ-Cytox Cell Viability Assay Kit, Daeillab Service Co., LTD.). (See 0.03 ml / min). In the perfusion culture chamber system according to the present invention, when the medium flow rate was slowly maintained at about 0.03 ml / min, cell viability was improved by about 110-115% compared to the static culture.

Example  17

Except that the culture chamber was subjected to ultrasonic biostimulation, the alveolar bone marrow-derived stem cells were cultured for about 4 days under the same conditions as in Example 14, and then the differentiation and growth of the cells were observed. The ultrasound was used for biostimulation at conditions of 50 mW / cm ² , 10% duty cycle, 10 minutes / day. In addition, as in Reference Example 1, the survival of the alveolar bone cells was analyzed using the WST-1 assay (EZ-Cytox Cell Viability Assay Kit, Daeillab Service Co., LTD.). (0.03 ml / min, 0.3 ml / min). In the perfusion culture chamber system according to the present invention, when the medium flow rate is slowly maintained at about 0.03-0.3 ml / min, the cell viability is improved compared to the static culture, and in particular, the cell viability is about 140-150 at 0.03 ml / min. % Improvement was made.

1: 4 gas mass flow meter 2: gas exchange device
3: culture chamber 4: sensor
5: reversed phase microscope 6: control medium vessel
7: sensor 8: fresh medium container
9: lung medium container 10: cell stimulator
11: load cell transmitter

Claims (15)

CO ₂ , O ₂ , N ₂ , 4-gas mixture control of air,
Control badge and
In a perfusion culture chamber system comprising a culture chamber,
The control medium container and the culture chamber are supplied with the 4-gas mixture from the 4-gas mixture control unit, the culture chamber is supplied with the medium in which the culture environment is controlled from the control medium container, and the cell culture of the culture chamber is Continuously monitored perfusion culture chamber system. The perfusion culture chamber system of claim 1, wherein the control medium container receives a medium from a fresh medium container. The perfusion culture chamber system of claim 1, wherein the medium discharged from the culture chamber is recovered to the control medium container. The perfusion culture chamber system of claim 1, wherein the culture chamber comprises a reversed phase microscope. The perfusion culture chamber system of claim 1, wherein the culture chamber comprises a cell stimulator. 6. The perfusion culture chamber system of claim 5, wherein the cell stimulus is ultrasonic stimulation. The perfusion culture chamber system of claim 1, wherein the control medium container comprises at least one sensor selected from the group consisting of a pH sensor, a DO sensor, an ammonia sensor, and a temperature sensor. The method of claim 1, wherein the culture chamber is a pH sensor, DO sensor, CO ₂ sensor, O ₂ A perfusion culture chamber system comprising at least one oath selected from the group consisting of sensors, temperature sensors, and humidity sensors. The perfusion culture chamber system of claim 1, wherein the cells to be cultured are adult stem cells. The perfusion culture chamber system of claim 1, wherein the culture chamber has a constant working volume. 7. The perfusion culture chamber system of claim 6, wherein the ultrasonic stimulation is at an intensity of 200 mW / cm ² or less. 7. The perfusion culture chamber system of claim 6, wherein the ultrasonic stimulation has a duty cycle of at least 5% and less than 50%. 7. The perfusion culture chamber system of claim 6, wherein the ultrasonic stimulation is a stimulation time in the range of 1 minute / day to 10 minutes / day. The perfusion culture chamber system of claim 6, wherein the ultrasonic stimulation is an intensity of 200 mW / cm ² or less, a duty cycle of 30% or less, and a stimulation time of 10 minutes / day. 15. The perfusion culture chamber system of claim 14, wherein the ultrasonic stimulation is 50 mW / cm ² intensity, 5% duty cycle, 10 minutes / day stimulation time.

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