TWI273135B - System and method for cultivating cells - Google Patents

Abstract

The present invention discloses a system for cultivating cells, wherein the system comprises a bioreactor, a pump, and an electromagnetic module comprising a coil and an electromagnetic stimulator. The cells are disposed within the bioreactor. The pump, connected to the bioreactor, is used to drive gas into the bioreactor, so as to ensure a sufficient gas supply for the cells. Additionally, the electromagnetic stimulator, connected to the coil, is used to provide a plurality of first signals, and the first signals are transported to the coil. Then the induced electromagnetic field is produced by the coil, whereby the induced electromagnetic field is applied on the cells within the bioreactor. Moreover, this invention also discloses the method for cultivating cells.

Description

1273135 % Nine, the invention relates to: [Technical Field] The present invention relates to a cell culture system, and more particularly to an in vitro cell culture system and method for integrating an electromagnetic field and a bioreactor. - [Prior Art] The combination of medicine and technology has made the field of medicine develop rapidly. Bone or fracture in the human body due to osteoporosis or accidental disasters • Unrecoverable, medically used electromagnetic fields to stimulate the bone cells of the human body to achieve a proliferative effect. There are many studies on the stimulation of bone cells in the body by electromagnetic fields, for example, in Tsai CL., Chang WH·, Liu TK, Wu KH, Additive effects of prostaglandin E2 and pulsed electromagnetic fields on fracture healing. Chinese Journal of Physiology. 2): 201-11, 1991·—The paper explores the mechanism of pulsed electromagnetic fields (PEMF) in stimulating osteoblasts in mice. n The bioreactor is also a well-known technique in which living cells of living organisms are placed in a reaction chamber, and the culture liquid and gas are supplied, and the in vitro cell culture is performed by the aeration principle, for example, in Liao CJ·, Chen. CF·, Chen JH·, Chiang SF, Lm YJ, Chang KY, Fabrication of porous biodegradable - polymer scaffolds using a solvent merging/particulate leaching method. Journal of Biomedical Materials Research. 59(4): 676-81, 20f Mar 15 · The method proposed in the text is. However, techniques for applying specific electromagnetic fields to bioreactors have not been disclosed. The use of a bioreactor for in vitro cell culture is simple, convenient, f-cost, and can be used to culture cells in large quantities in vitro, and the conditions for implementation are easily controlled. If the electromagnetic field can be combined with the bioreactor, another kind of 6 8 1273135 "d: diterpene treatment or culture will be produced. Therefore, the cells of the present invention establish bone formation in a new environment in vitro, which is easy, rapid and observable. Differentiation and more tolerance [Summary of the Invention] In view of the above-mentioned background of the invention #, for the purpose of providing a clear _ _ pure and = ~ requirements, the body of the reactor & - integrated electromagnetic field and biological proliferation - a kind of scorpion bone The cell (4) the extracellular cell culture secret contains - the biological reaction ϊ (4) has the coil and the electromagnetic field stimulator electromagnetic field module. In the bioreactor described above, and the above pumping system connects the bio-driven gas to the bioreactor In order to supply sufficient cells, iJU describes the electromagnetic field stimulator connected to the coil. The electromagnetic field stimulates r production, hits the first position and transmits a plurality of first signals to the above coil to generate an induced electromagnetic field. The stimulus is located in the above-mentioned biological reaction crying cells. Preferably, the above system further comprises a three-dimensional structure of the scaffolds placed in the bioreactor. To support the cells to be cultured. Preferably, the system further includes a timer connected to the pump to facilitate timing driving of the gas into the bioreactor. Preferably, the bioreactor described above is located in the coil. Preferably, the above system further comprises a sensor for detecting the electric field strength of the induced electromagnetic field of 1273135 to generate a plurality of flutes. The second signal to the electromagnetic field of the above-mentioned electromagnetic field 3 and f feeds a plurality of sizes. In the case of adjusting the plurality of first signals, the inductive electromagnetic field is periodically applied to the cells to be cultured. It has been confirmed by static observation that the root-seeding method is for osteoblasts === cell culture system and side [Embodiment]

Si its composition. Obviously, the practice of the invention is not limited to the cell it. On the other hand, _ know == (four) to avoid causing this (four) unnecessary _. The invention will be described in detail below, and in addition to these detailed descriptions, 3 is widely practiced in other implementations, and the scope of the invention is not limited, and the scope of the following patents will prevail. In the tea test A-A and B-B, an embodiment of the present invention discloses an in vitro cell culture system 10, particularly for the culture/manufacturing of osteoblasts, which comprises a bioreactor 12, a group of The pump 14 and an electromagnetic field module having a coil 16 and an electromagnetic field stimulator 18. The bioreactor 12 is a closed device or container for arranging cells η and broth 2, and the like. In a preferred embodiment of the present embodiment, the in vitro cell culture system further comprises a plurality of cell supports located within the bioreactor 12 to provide cell attachment growth. The above-mentioned cell scaffolds may be porous or fiber-interwoven structures, the material of which is a biodegradable material or a material having biocompatibility. Preferably, the shape of the cell scaffold is disc-shaped so as to be stacked on each other to form a coaxial stack. The % Pu 14 is connected to the bioreactor 12 to drive the gas into the bioreactor η '^ to supply enough gas to the cells 11'. This embodiment takes the use of the 1273135 gas principle to culture osteoblasts. Preferably, system 10 further includes a timer 15 coupled to pump 14 for timingly driving the gas to bioreactor 12, where the gas is preferably selected to be a gas having an oxygen component. In the present embodiment, the coil 16 is used to generate an induced electromagnetic field, so that the cells 11 in the biological reaction 12 receive a uniform electromagnetic field stimulation, wherein the coil lj further includes a solenoid. In another preferred embodiment of the embodiment, the bioreactor 12 is located in the coil. The electromagnetic field stimulator 18 is connected to the coil 16, and the electromagnetic field stimulator 18 can generate a plurality of first signals 17 and transmit a plurality of first-signals 17 to the coils 16 to generate a magnetic field, whereby the stimulus is located above. Cell n in bioreactor 12. When the shape of the plurality of cell holders described above is disc-shaped and forms a coaxial stack, the direction of the magnetic field of the induced electromagnetic field is perpendicular to the surface of the cell holder. In addition, the electric field stimulator 18 can adjust the waveform and the intensity of the supply of the first signal 17, to determine the frequency of the induced electromagnetic field, the wave type and intensity of the ship, and thereby achieve the effect of stimulating the cell 11, wherein the induced electromagnetic field here Also known as "stimulation, (Stmmlatlon)" on cells. In general, the induced electromagnetic field is low frequency and low intensity. The bioreactor 12 is positioned 3 degrees with the sensor 19 to generate a plurality of second signals and feedback a plurality of electromagnetic field stimulators 18 to accurately control or adjust the electric power applied to the cells 11. The field is in the desired size range. ! The type of signal - 17 can be a single pulse (call p/iei =, bum). In a further preferred embodiment of the present embodiment, J 7, Hz 133 ms, ^ ^ MOmV/cm. The electrical enthalpy is about to increase the amount of osteoblasts using the system 10. The pulsed type of lightning is selected to be 3, and the magnetic field of different ages is stimulated by the magnetic field of Pulsed electromagnetic 1273135 1 to stimulate the osteoblasts; the change of activity of the enzyme (Alkaune Phosphatase; ALP) is observed. === % has an electric field strength of 4 mV/cm. For example, in the third A罔2 privately stimulated electromagnetic field, the activity of the element is in the time group, and the curve 102 is stimulated by the electromagnetic field for 2 hours every day, and the activity of the activity is changed with time. According to the third two, in: t匕 control, group height, which has more _ osteoblasts than the other two groups = ^ choose the brother B is not the electric field that will stimulate the electromagnetic field to SmV / cm The intensity is observed in the change of the activity of __, =:, magnetic = __ acid enzyme activity versus time =, as = enzyme activity versus time. According to the eighth: after the magnetic field strength, miscellaneous The activity of the enzyme has a distinct mineralization phenomenon, and the mineralization phenomenon is more obvious. From the third a map and the id, the Japanese singer, the spring is not, under the stimulation of different electromagnetic fields, the stimulation of 2 per day That is, there is a differentiation situation of _. The brush is a chemical effect, and the change of the two-week nutrient solution is observed. In the experiment, the cell proliferation is carried out to prove that the stone is placed on the stone. The fourth A picture and the fourth B picture are in the culture medium under the stimulation of the electromagnetic field every two weeks. (4) _^Γ) ϋ Ϊ 273135 Degree change graph. As shown in Figure 4A, the excited electromagnetic field has a field strength of 4 mV/cm, and the curve 200 is the change of glucose concentration in h in the culture medium without electromagnetic field stimulation as a control group, and the curve is carried out every day under electromagnetic field stimulation for 2 hours. The change of glucose concentration in the culture medium with time, the curve, the field _ hour under the culture solution _ sugar concentration versus time === The concentration of glucose in the broth of the four A culture medium shows a trend of = for the different electromagnetic field stimulation 'where' The electromagnetic field was made in the 8-hour group. The ΜίΪΐΐϋ week was lower than the control group, indicating that it needed more glucose = enough (4). The fourth diagram shows the change of the glucose concentration in the culture solution by increasing the electromagnetic field excited to 8 mV/cm. The concentration of glucose in the culture solution under the absence of electromagnetic field is different in the curve 212. The distribution of the sugar wave in the 2 small-inch mouth-washing liquid at different times, the curve 214 ί 8 hours under the culture medium, the concentration of glucose in the culture solution at different times == according to the younger brother, the culture medium (four) sugar concentration After being divided into 2 and 8 hours of stimulation per $ stone field, it was obviously in a decreasing state, and the glucose concentration in the medium of 352 hours in the electromagnetic field was low, indicating that it needs more _ Sugar to produce enough energy ^ according to the younger izgA map and the fourth pass, curves 212 and 21 and 204 show a decreasing change, therefore, shown in this culture === osteoblasts with the extension of the correction, the required _ sugar It also increases, and the energy required by the brother increases, which also implies that the cells have a good increase. The table then looks at the growth of the cell from its generation. In the fifth and second weeks, the electromagnetic field is stimulated by electromagnetic fields of different hours every day. Therefore, the electromagnetic field has the electric field intensity of m recorded, and the curve: j=the concentration of lactic acid in the culture solution is changed according to time, as the control of sputum replenishment every day under electromagnetic field stimulation for 2 hours. The milk in the culture solution; the ice 302 is systemized, and the curve 304 is stimulated by the electromagnetic field every day, and the time is 1273135: day =,. On the other hand, the red B _ will be stimulated by the irm lion New Zealand bribes to touch the towel Wei concentration ί : no electromagnetic field stimulation of the culture medium in the culture of the lactate infiltration time as a control group, curve 312 line per day under electromagnetic field stimulation for 2 hours ^ Nutrient = Gravity Concentration vs. Time, Curve 314 is a daily change in the concentration of lactic acid per day under electromagnetic pressure for 8 hours. According to the fifth a map = five B map, as time increases, the concentration of lactic acid in the culture solution also increases, indicating that the metabolic limbs of the cells are stable' also implies that the integrated bioreactor and the culture of the magnetic flux are suitable. Osteoblasts grow. The piu_electromagnetic field (PEMF) stimulation is integrated in the bioreactor, and it can be confirmed by experiments that it can be the proliferation and differentiation of the cells, and the new ones of the osteogenesis of the osteoblasts in the body can be cultured. The invention discloses an in vitro cell culture system comprising an electromagnet module of a bioreactor, a pump and a coil and an electromagnetic field. The cell line is disposed in the bioreactor described above, and the pumping system is coupled to the bioreactor to drive a gas into the bioreactor to supply a sufficient amount of gas to the cells. In addition, the electromagnetic field stimulator is connected to a line, and the electromagnetic field stimulator can generate a plurality of first signals and transmit a plurality of first signals to the coils to generate an induced electromagnetic field, thereby stimulating the bioreactor located above. The cells in it. In another aspect, the invention also discloses a method of culturing sputum cells. And obviously, the invention may have many modifications and differences, as described in the above examples. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced in other embodiments. The above are only the preferred embodiments of the present invention, and are not intended to limit the scope of the claims of the present invention; all other equivalent changes or modifications which are not departing from the spirit of the present invention should be included in the following claims. Fan ^ 1273135 within 0

1273135 [Simple description of the system] System ^AH® mixed age Wei Wei, - the out-of-plane cell culture system touched the county Weizhi, -_The second figure provided by the axial culture = 5 «(4) Medium electromagnetic field stimulator

The change of activity; the observed observation of the cell (4) acid enzyme in two weeks, the induced electric field intensity of 8mm, the daily excitation of the bone butterfly _ alkali (four) acid enzyme _ _, the induced electric field strength 4 mV / em Observed under the ship every day _ culture liquid towel glucose concentration fourth B picture in two weeks, the induced electric field strength 8 mV / cm, the glucose concentration of the contact culture liquid towel observed under different electromagnetic fields per day Change chart; Figure 5A shows the change of lactic acid concentration in the culture medium observed by electromagnetic field with different hours in two weeks, the induced electric field intensity is 4mV/cmT; During the week, the induced electric field intensity is 8 mV/cin, and the change of the lactic acid concentration in the culture solution observed under the electromagnetic field stimulation of different hours every day. 10 1273135 [Explanation of main component symbols] 10 In vitro cell culture system 11 Cell 12 Bioreactor 13 Cell scaffold 14 Pump 15 Timer 16 Coil 17 First signal

18 Electromagnetic field stimulator 20 Culture medium 19 Sensor 100 without electromagnetic field stimulation in the environment 102 daily electromagnetic field stimulation 2 small curve = scallop acid activity change curve τ environmental towel although __ activity variable 104 daily electromagnetic field stimulation 8 hours of the curve 110 alkaline phosphatase activity in the environment without electromagnetic field stimulation

114 alkaline phosphatase activity in the environment of 8 hours of electromagnetic field stimulation every day 202 204 210 212 214 Wei electric stone % 刺激 刺激 环境 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 每天 电 电 电 电 电 电 电 电 电 电 电 电 电 电 电 电 电Ff sugar concentration curve without electromagnetic field stimulation in the environment, the glucose concentration curve of glucose in the environment is stimulated by electromagnetic field every day 2: two, the degree of change curve is stimulated by electromagnetic field every day, 8 small disaccharide sugar concentration curve) curve

D 15 1273135 300 302 304 310 312 314 Change in lactic acid concentration in the environment of each 夭:: The lactic acid concentration change curve in the daily environment. The lactic acid concentration change curve in the % of the lactic acid concentration curve is stimulated by the electromagnetic field every day.

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Claims (1)

1273135 Announcement 10, the scope of patent application: 1. An in vitro cell culture system, the in vitro cell culture system comprises: a bioreactor for arranging cells; a pump connected to the bioreactor to drive a gas into the a coil for generating an inductive electromagnetic field, wherein the inductive electromagnetic field is used to stimulate cells located in the bioreactor; An electromagnetic field stimulator connected to the coil, wherein the electromagnetic field stimulator can generate a plurality of first signals and transmit a plurality of the first signals to the coil, and generate the induced electromagnetic field, thereby stimulating the bioreactor Cell. 2. The in vitro cell culture system of claim </ RTI> wherein said cells comprise osteoblasts. 3. If you apply for a patent scope! An in vitro cell culture system wherein the gas described above has an oxygen component. 4. The in vitro cell culture system of claim 1, further comprising a plurality of cell scaffolds having a two-dimensional structure, the plurality of cell scaffolds being located in the bioreactor to provide cell attachment growth. 5. The in vitro cell culture system of claim 4, wherein the plurality of cell scaffolds are in the shape of a disk. 6. The in vitro cell culture system of claim 5, wherein the plurality of cell supports are stacked one on another to form a coaxial stack. 7. The in vitro cell culture system of claim 6, wherein the magnetic field direction of the induced electromagnetic field is perpendicular to the surface of the plurality of cell scaffolds. ^8. The in vitro cell culture system of claim 1 of the patent scope, further comprising a juicer connected to the pump, and driving the gas into the bioreactor at a timing. 9. The in vitro cell culture system of claim 1, wherein the coil comprises a solenoid. 10. The in vitro cell culture system of claim 1, wherein the organism 8 1273135 reactor is located in the coil. 11. The in vitro cell culture system of claim 3, wherein the frequency of the first signal is about 7.5 Hz. 12. The in vitro cell culture system of claim 1, wherein the first signal of the above-mentioned teachings is a single pulse. 13. The in vitro cell culture system of claim 12, wherein the plurality of first signals have a period of about 133 ms and a single pulse width of about 〇. 4. The in vitro cell culture system of claim 3, wherein the plurality of first signals are in the form of a sputum. 15. The in vitro cell culture system of claim i, wherein the electromagnetic field has an electric field strength ranging from about i to 10 mV/cm. ^ 1δ·如 = Please refer to the in vitro cell culture system of the scope of the patent item i, wherein the above-mentioned plurality of signals are provided for less than or equal to 8 hours per day. 17. The in vitro cell culture system of claim 1 , further comprising a sensor for detecting an electric field strength of the induced electromagnetic field to generate a plurality of second signals and feeding back the plurality of second signals to the electromagnetic field stimulator, Thereby, the size of the plurality of the first signals is adjusted. 18--in vitro cell culture method, the in vitro cell culture method Xiao Ku · Place the cells in a bioreactor; · The reaction reactor is driven by the pump of the bioreactor to drive a gas into the biological anti-inr and An induced electromagnetic field is generated 'by which the stimulus is located in the life. 19. The osteogenic cells are included in the scope of the patent application. The in vitro cell culture method of the 18th aspect, wherein the cell is as described in the patent application. The gas culture method of the invention has an oxygen component. 21. The in vitro cell culture system of claim 18 is attached to the cell culture system. A plurality of cells having a three-dimensional structure 18 1273135 read cell scaffolds are located in the bioreactor. 22. The in vitro cell culture method according to claim 21, wherein the plurality of cell scaffolds are in the shape of a disk. 23. The in vitro cell culture method of claim 22, wherein the plurality of cell scaffolds are stacked one on another to form a coaxial stack. 24. The in vitro cell culture method according to claim 23, wherein the magnetic field direction of the induced electromagnetic field is perpendicular to a surface of the plurality of cell scaffolds. 25. The in vitro cell culture method of claim 18, wherein the gas is delivered to the bioreactor at a time by a timer connected to the pump. 26. The in vitro cell culture method according to claim 18, wherein the electric field strength of the upper electromagnetic field is in the range of about 1 to 10 μm/cm. 〜27. The in vitro cell culture method of claim 18, wherein the field module comprises: a coil; and an electromagnetic field stimulator connected to the coil, wherein the electromagnetic field stimulator can generate a plurality of first The signal transmits a plurality of the first signals to the coil to generate the induced electromagnetic field. 28. The in vitro cell culture method of claim 27, wherein the above line comprises a coil. 29. The in vitro cell culture method of claim 27, wherein the bioreactor is located in the coil. 30. The in vitro cell culture method of claim 27, wherein the first signal has a frequency of about 7.5 Hz.夂 31· In the in vitro cell culture method of claim 27, wherein the first signal type is a single pulse. 32. The in vitro cell culture method according to claim 31, wherein the first signal has a period of about 133 ms and a single pulse width of about 〇·3 ms. 33. The in vitro cell culture method of claim 27, wherein the first signal is in the form of a pulse train. 19 % 1273135 34. The in vitro cell culture method according to claim 27, wherein the plurality of first signals are provided for less than or equal to 8 hours per day. 35. The in vitro cell culture method of claim 27, further comprising a feedback program, the feedback program comprising: detecting an electric field strength of the induced electromagnetic field by a sensor, and generating a plurality of second signals; A plurality of the second signals are fed back to the electromagnetic field stimulator, thereby adjusting a size of the plurality of the first signals. 20 (D