WO2012077980A2

WO2012077980A2 - Stem cell culturing apparatus

Info

Publication number: WO2012077980A2
Application number: PCT/KR2011/009440
Authority: WO
Inventors: 황해령; 글렌 칼더헤드로버트
Original assignee: (주)루트로닉
Priority date: 2010-12-07
Filing date: 2011-12-07
Publication date: 2012-06-14
Also published as: KR101270133B1; WO2012077980A3; KR20120063154A

Abstract

The present invention relates to a stem cell culturing apparatus which uses light of a specific wavelength to increase the proliferation rate and life of a stem cell. The stem cell culturing apparatus of the present invention comprises: a sealed culturing chamber which accommodates therein a vessel containing a stem cell to be cultured, and which has a transparent partition wall in an upper portion thereof; gas adjusting means for adjusting an internal environment of the culturing chamber; and a light source arranged outside the culturing chamber to irradiate light to the inside of the culturing chamber through the transparent partition wall.

Description

Stem Cell Culture Device

The present invention relates to a stem cell culture apparatus, and in particular, in the visible light region to increase the proliferation rate and lifespan of the stem cells using a wavelength of the wavelength of the near-infrared region to separate the light source and the culture environment, It is about a stem cell culture apparatus that is not affected by the temperature.

Stem cells are immature cells that have not yet differentiated and are cells that have the capacity to divide and replicate indefinitely while maintaining an undifferentiated state in in vitro culture. In addition, it is a parent cell capable of differentiating into many different kinds of cells that make up an organism depending on the time of development and location of the individual. Extracting these stem cells and transplanting them into the patient's diseased area restores the damaged cells to normal, healing the disease or regenerating tissue and organs.

Stem cell research requires a sufficient amount of stem cells, but there are practical problems to gather enough cells. In addition, stem cells have a problem that can face serious ethical problems, depending on their origin.

In humans, stem cells are pluripotent stem cells that form when the fertilized egg first divides (up to 8 cell stages after fertilization of eggs and sperm), and embryonic stem cells that are produced by continuous division of these pluripotent stem cells. It can be divided into cells, pluripotent cells, and multifunctional stem cells (adult stem cells, multipotent cells) contained in mature tissues and organs.

Embryonic stem cells have caused considerable controversy and some countries have banned the use of embryonic stem cells. Adult stem cells are collected from a patient and transplanted into the patient, also known as autologous stem cells, and virtually no prohibition problem. Successful use of autologous platelet rich plasma (PRP) for wound and bone treatment involves stem cells isolated from PRP.

However, the number of autologous stem cells is very small and it is difficult to proliferate in sufficient amounts for the main procedure. Exogenous stem cells derived from outside the patient can be secured in a sufficiently larger number than autologous stem cells. Ethically acceptable sources of external origin include human organs and tissues, cord blood, animal embryonic stem cells (not controversial), and human amniotic fluid donated to specific stem cell banking institutions. The latter provides abundant multifunctional stem cells and has been approved by the Vatican for various stem cell sources.

The inventors of the present invention have found that when the stem cells are regularly irradiated with light of a specific wavelength band, the proliferation rate and lifespan of the stem cells are significantly increased. At this time, in order for the light to affect the proliferation and life of the stem cells, the amount of light must be provided to the stem cells. Therefore, the distance between the stem cells and the light source should be close, but at the same time, the output of the light source should be high enough. A problem has been found that directly affects.

It is therefore an object of the present invention to provide a stem cell culture apparatus for increasing the proliferation rate and life span of stem cells using light of a specific wavelength band.

Another object of the present invention is to remove a stem cell culture apparatus from which factors that may affect the culture environment of stem cells by heat generation of a light source.

The object of the present invention is a hermetic culture chamber for receiving a container containing the stem cells to be cultured therein, a light source control unit for controlling the light source and the light source for irradiating light to the container and located on the culture chamber, the light source control unit It is achieved by a stem cell culture apparatus characterized by having a proliferation mode of stem cells or a maintenance mode of stem cells.

In the stem cell culture apparatus according to the present invention, it is preferable to further include a temperature control means for controlling the temperature inside the culture chamber.

In addition, the stem cell culture apparatus according to the present invention may further include a gas control means for adjusting the gas concentration in the culture chamber, wherein the gas concentration is preferably determined as the ratio of O2 and CO2.

In the stem cell culture apparatus according to the present invention, the light source is preferably a light emitting diode (LED) or a laser diode (LD). In this case, it is preferable to defocus when using the laser diode LD. The wavelength of the light irradiated from the light source is preferably in the range of 400 nm to 940 nm, and in particular, the energy of the light irradiated from the light source to the container is preferably 1 to 15 J / cm 2.

In the stem cell culture apparatus according to the present invention, the propagation mode of the light source control unit is a control mode of operating the light source once to two times in two weeks, and the maintenance mode is a control mode of operating one to five times in two months.

In the stem cell culture apparatus according to the present invention, it is preferable to further include a light source temperature control means for controlling the temperature of the light source. At this time, the light source temperature control means is an air-cooled or air-cooled refrigerant tank including a cooling fan, one end is connected to the refrigerant tank inlet hose for introducing the refrigerant, one end is connected to the refrigerant tank outflow hose and inlet hose for outflow of the refrigerant And connected to the other end of the outlet hose may be a water cooling including a refrigerant circulation for cooling and circulating the refrigerant.

In the stem cell culture apparatus according to the present invention, it is preferable to further include a distance adjusting means for adjusting the height of the light source up and down so as to adjust the distance between the container and the light source.

In the stem cell culture apparatus according to the present invention, the culture chamber further includes a light-transparent transparent partition on the upper portion, and the light source preferably irradiates light into the culture chamber through the transparent partition.

In the stem cell culture apparatus according to the present invention preferably further comprises a light blocking member which is a cover for blocking external light to the light source and the transparent partition wall, wherein the light blocking member is air for circulating the air inside the sealing means with the outside It may further include a circulation means.

Cultivating stem cells using the stem cell culture apparatus according to the present invention has an effect of significantly increasing the proliferation rate and lifespan of the stem cells, in particular to effectively prevent the influence of the light source on the temperature, which is an important factor in the culture of stem cells There is an advantage.

1 is a conceptual diagram of a stem cell culture apparatus according to an embodiment of the present invention,

Figure 2 is a conceptual diagram of a light source and a light source temperature control means of the stem cell culture apparatus according to an embodiment of the present invention,

Figure 3 is a cross-sectional view of the refrigerant tank of the stem cell culture apparatus according to an embodiment of the present invention.

The terms or words used in this specification and claims are not to be construed as being limited to their ordinary or dictionary meanings, and the inventors may appropriately define the concept of terms in order to best describe their invention. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that the present invention.

Therefore, the embodiments described in the specification and the drawings shown in the drawings are only the most preferred embodiment of the present invention and do not represent all of the technical idea of the present invention, various modifications that can be replaced at the time of the present application It should be understood that there may be equivalents and variations.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a conceptual diagram showing the inside of the stem cell culture apparatus according to an embodiment of the present invention.

Stem cell culture apparatus of this embodiment is composed of a culture chamber 100, gas control means 200, temperature control means (not shown), the light source 300, the light source temperature control means (310).

The culture chamber 100 is not shown as a structure for receiving the container 10 containing the stem cells therein, but the door 10 is provided with a door that can be inserted into or removed from the culture chamber 100, the culture chamber 100 Windows can be installed to observe the inside. When the window is installed, it is preferable that a curtain or the like is provided to cover the window so that external light does not flow inside when it is not observed. Such doors and windows may be integrally formed.

The container 10 accommodated in the culture chamber 100 may be a test tube, a flask, a culture dish. Stem cells accommodated and cultured in the container 10 may be stem cells of all types of humans and animals, such as neuroepithelial stem cells, mesenchymal stem cells, hematopoietic stem cells.

The culture chamber 100 is coupled to the gas control means 200 that can appropriately control the gas concentration in the air in the culture chamber 100 to the stem cell culture to provide an environment such as a temperature suitable for stem cell culture do. In particular, the gas control means 200 needs to include an appropriate O 2 / CO 2 ratio in the culture of stem cells and gas circulation means for maintaining such a gas atmosphere. The gas circulation means preferably includes an inlet for introducing a gas having an appropriate O 2 / CO 2 ratio into the culture chamber 100 and an outlet for discharging the gas inside the culture chamber 100.

In addition, the culture chamber 100 is coupled to a temperature control means (not shown) for adjusting the internal temperature of the culture chamber 100, the temperature control means (not shown) is a direct combination of the heating means and cooling means as a configuration Combined to heat or cool the inner wall of the chamber 100 or indirectly to control the temperature of the culture chamber 100 by heating or cooling the gas introduced through the gas control means 200 to enter the culture chamber 100 Can be configured.

The culture chamber 100 also has a temperature sensor or gas sensor that can confirm whether the gas control means 200 is properly adjusting the culture environment and the culture chamber 100 that calculates the data of these sensors and displays them outside the culture chamber 100. A control unit may be added, and such a culture chamber 100 may further include a light source 200, a temperature control unit (not shown), a gas control unit 200, and a distance control unit, in addition to simply displaying data of a sensor. 320 and the like can be configured to control.

In order to control the control unit in a state in which the user is separated from the inside of the culture chamber 100, the control panel of the control unit is located outside the culture chamber 100, and the control panel controls the light irradiation time and light irradiation operation set by the user. Includes control buttons for adjusting parameters such as remaining time, LED output (mW), light output characteristics (continuous wave or pulse frequency setting), light wavelength, and a display that outputs the adjusted results.

The upper portion of the culture chamber 100 includes a transparent transparent partition wall 110, and the upper portion of the transparent partition wall 110 includes a light source 300 for irradiating light adjusted to the inside of the culture chamber 100. The transparent transparent partition wall 110 may use a glass panel in general, but may use an optical filter capable of blocking light of a specific wavelength depending on the purpose. The transparent partition wall 110 passes light but blocks air or heat to block the temperature inside the culture chamber 100 from being affected by the heat of the light source 300.

The light source 300 is preferably a surface light source using an array of light emitting diodes or laser diodes, and the wavelength band is selected from the visible region in the near infrared range of 400 nm to 940 nm, and the range of the wavelength band is narrow (1 nm or less). It is preferable. When using a laser semiconductor array, each laser semiconductor is defocused so that uniform light is irradiated to the container. In addition, it is preferable that the light source 300 can adjust the light wavelength range, and the adjustment of the light wavelength range may be implemented by replacing the diode head or by arranging light emitting diodes having different emission wavelength characteristics in an array.

In order to adjust the light output of the light source 300 or its period, the continuous or pulse width of the japan, a separate light source control unit may be provided outside. In this case, the light source control unit may be integrally formed with the control unit of the culture chamber 100. Preferably, the light source controller precisely adjusts the wavelength, energy level, and frequency of the light.

In particular, the light source control unit is preferably such that the energy of the light reaching the container by the light output is 1 to 15 J / cm 2. In addition, the light source control unit may have a proliferation mode for proliferating and a maintenance mode for maintaining the proliferated stem cells. The propagation mode is a control mode for operating the light source once to seven times in two weeks, and the maintenance mode is a control mode for operating one to five times every two months.

On the other hand, the light source 300 is coupled to the light source temperature control means 310 for cooling the heat of the light source (300). When the light emitting diode 300 is a semiconductor, heat generation is insignificant, but heat generation of the driving circuit of the light emitting diode is transmitted to the light emitting diode, which affects its operating characteristics, thereby shifting the wavelength of light from the ideal wavelength band. When the light source 300 is a semiconductor laser, both the semiconductor laser and the heat generation of the circuit should be considered. The light source temperature regulating means 310 removes the heat of the driving circuit so that the light emitting diode can irradiate light of an ideal wavelength band.

The light source temperature regulating means 310 may use various cooling means such as an air cooling method in which a cooling fan is coupled to the opposite side of the light irradiation surface of the light source 300 or a water cooling method by circulation of the refrigerant.

2 is an exemplary structure when the light source temperature control means 310 is water-cooled. Basically, the light source temperature control unit 310 includes a refrigerant tank 312, an inlet hose 316, an outlet hose 317, and a refrigerant circulation unit 318. The coolant tank is coupled to a surface opposite to the light irradiation surface of the light source 300 to allow heat generated in the driving circuit of the light source 300 to escape to the coolant stored in the coolant tank. The refrigerant tank 312 includes an inlet 314 and an outlet 315 for inflow of refrigerant into the refrigerant tank 312, and an inlet hose 316 and an outlet for the inlet 314 and the outlet 315, respectively. Hose 317 is coupled. Refrigerant of the refrigerant tank is introduced and discharged into the refrigerant tank 312 by the inlet hose 316 and the outlet hose 317 connected to the refrigerant tank, respectively, and the other end of the inlet hose 316 and the outlet hose 317 The refrigerant circulation part 318 is connected. The refrigerant circulation part 318 receives the refrigerant from the outflow hose 317 and cools it, and is configured to discharge the cooled refrigerant back to the inlet hose 316 at a constant pressure so that the refrigerant receives heat from the refrigerant tank 312 as a whole. The refrigerant is circulated while releasing heat from the circulation unit 318.

3 is a cross-sectional view of the refrigerant tank, the refrigerant tank 312 may be a tank shape having a generally empty space for storing the refrigerant, but as shown in FIG. It can have a structure that can be delivered to. In addition, between the light source 300 and the coolant tank 312 to facilitate the heat transfer from the light source 300 to the coolant in the coolant tank 312, a heat transfer member 311, which is a plate made of a material having a high heat transfer rate such as a metal material, is provided. Can be formed.

In addition, the light source 300 is coupled to the distance adjusting means 320 for adjusting the light irradiation height of the light source 300 is configured to adjust the distance between the stem cells and the light source 300. The light source 300 is supported by the support 321 horizontally supported, the distance adjusting means 320 is coupled to the vertical rod (rod) such that the support 321 is movable up and down and the light source 300 and the stem Adjust the distance to the container 100 containing the cells. At this time, the control of the distance adjusting means 320 is preferably configured to be controlled by the light source control unit or the culture chamber 100 control unit.

On the other hand, in order to prevent the external light that is not irradiated from the light source 300 is introduced into the culture chamber 100 through the transparent partition wall 110 and surrounds the transparent partition wall 110 and the light source 300 and the external light. Light blocking member 330 to block may be installed. In particular, when the light source temperature control means 310 is air-cooled, the air does not escape to the outside by the light blocking member 330 and thus the light source 300 may not be cooled even though the light source temperature control means 310 is present. have. To this end, the air circulation means 340 capable of circulating the internal air and the outside air of the light blocking member 330 is preferably installed in the light blocking member 330, the air circulation means 340 is to be a simple ventilation hole It can be but consists of a fan that actively introduces or discharges air.

Experimental Example

Pig tissue is a good example of biocompatibility in humans. For example, specially treated pig skin is commonly used for transplantation treatment of human burns, and pig heart valves are used as replacements for human heart valves.

With regard to stem cells, the best cell for research is pluripotent stem cells from embryos. Because the source contains a variety of fragments, the number of embryos available for each animal is significant, and neuroepithelial stem cells (NSCs) are relatively easy to obtain from the embryonic midbrain conduit. Neuroepithelial stem cells are genetically destined to differentiate into many types of neural tissues, especially neurons and astrocytes.

Pig neuronal epithelial stem cells were grown in culture, and experiments were performed to maintain the number of cultured neuroepithelial stem cells for a certain period.

1. Preparation for Experiment

Mesencephalic plates were separated from embryos of 14-day-old pigs, and neuroepithelial stem cells were extracted under a microscope. For further experiments to verify that proliferated stem cells can be properly transplanted into other animals, pigs were able to identify host tissues and donor tissues using transgenic 'Green Pigs'. The extracted cells were cultured in a petri dish containing 15% Fetal Bovine Serum and a culture solution containing antibiotics. The cells were incubated at 37 ° C. and the formation of neurospheres was observed. The neurospheres formed were confirmed to contain true stem cells by Nestin immunostaining.

2. Growth experiment

After the formation of the neurosphere, the culture dish was irradiated with a defocused laser beam of 830 nm. GaAlAs diode laser (Model: OhLaser-3D1, JMLL Co., Ltd.) was used as the laser beam generator. Irradiated about 3 times every 2 weeks, but separated the experimental group 1, 2, 4, 6, 8, 10 and 15 J respectively. The energy of / cm 2 was investigated. The laser beam generating means was placed at exactly the same height from the culture dish so that the defocused laser beam was 5 cm in diameter in the culture dish. Each experimental group was treated in exactly the same way, and a control group without irradiating a laser beam was prepared for comparison with the experimental group.

The proliferation of neurospheres was evaluated 1, 2, 4 and 6 weeks after light irradiation, and the evaluation method was used to evaluate whether the neural spheres contained stem cells by using Nestin staining on the sample neurospheres. .

As a result of the proliferation experiment, it was confirmed that the number of neurospheres was increased as the energy of the laser beam increased from 10 J / cm 2 without the laser beam irradiation. All propagated neurospheres were positively stained with good numbers of viable stem cells. In the sample irradiated with a laser beam of 15 J / cm 2, the number of neurospheres decreased drastically but still remained higher than that of the control group. Especially, after 6 weeks of evaluation, the samples irradiated with 6, 8, 10J / cm2 laser beams had statistical significance (p (parent ratio) = 0.05, 0.01, 0.001), especially 10J / cm2 laser beams It appeared to be most suitable for proliferation.

3. Maintenance experiment

While maintaining the propagated neurosphere cultures in an incubator, a laser beam defocused at 830 nm of continuous wave once or twice a month was irradiated with an energy of 10 J / cm 2 which was found to be most effective in the propagation experiment. Meanwhile, a control group not irradiated with a laser beam was prepared for contrast with the experimental group. It was confirmed whether the number of neurospheres is maintained at 1, 2, 4, 6, 8, 12 months after the start of the experiment, and the amount and quality of stem cells by applying Nestin staining to the sample neurospheres.

As a result, the number of neurospheres was maintained with statistical significance in both the experimental group irradiated with the laser beam once and the experimental group irradiated twice with the control group until 12 months. The quality and quantity of stem cells of each neurosphere were also maintained. In the control group, the number of neurospheres began to decrease on the second month of evaluation, and after 12 months, the neurospheres remained few and the quality and quantity of stem cells were very low, and some of them were differentiated. Irradiation of the laser beam once a month was evaluated as being most suitable for the maintenance of propagated neurospheres.

4. Transplant Experiment

A 12-week-old male mouse was used to carry out transplantation of neurospheres of pigs cultured above. Both mice had Parkinson's disease and Huntingdon's disease, and were divided into two groups, a transplanted group and a non-grafted group. For the experimental group, neurospheres cultured in the substantia nigra were transplanted.

In the experimental group, some samples were dissected every two weeks to confirm the survival of donor tissue by checking green fluorescent material by ultraviolet irradiation, and stem cell survival was confirmed by nestin staining.

In addition, the specimens were stained to determine whether the nervous system (Golgi stain) or astrocytes (Holzer stain) are growing in the transplant area. At the same time, it was also confirmed that the disease-related behavior was improved.

Experimental results showed that the Parkinson's and Huntingdon's groups had improved gait for 6 months from 6 weeks after transplantation. In particular, the incidence of in situ in the Parkinson's disease group was reduced, and memory in the Huntingdon's group was improved (Maze Training). In the histological experiments of the Parkinson's disease group, new dopaminergic neurons (Golgi staining) grew in the ventral epidermal region of the midbrain, and the nervous system grew. It was confirmed. In the Huntingdon group, neuronal cells (Holzer staining) showing green fluorescence were grown.

Claims

An airtight culture chamber containing a container containing stem cells to be cultured therein;

Located above the culture chamber and includes a light source for irradiating light to the vessel and a light source control unit for controlling the light source,

The light source control unit stem cell culture apparatus characterized in that it has any one or more of the proliferation mode of the stem cells or the maintenance mode of the stem cells.
The method of claim 1,

Stem cell culture apparatus further comprises a temperature control means for adjusting the temperature inside the culture chamber.
The method of claim 1,

Stem cell culture apparatus further comprises a gas control means for adjusting the gas concentration in the culture chamber.
The method of claim 3, wherein

The gas concentration is stem cell culture apparatus, characterized in that the ratio of O2 and CO2.
The method of claim 1,

The light source is a stem cell culture device, characterized in that using a light emitting diode (LED) or a laser diode (LD).
The method of claim 1,

Stem cell culture apparatus characterized in that the wavelength of the light irradiated from the light source is in the range of 400nm to 940nm.
The method of claim 1,

The energy of the light irradiated from the light source to the container is a stem cell culture device, characterized in that 1 to 15J / cm2.
The method of claim 1,

The proliferation mode is a stem cell culture device, characterized in that for operating the light source once every two to seven times.
The method of claim 1,

The maintenance mode is a stem cell culture device, characterized in that for operation once every five months.
The method of claim 1,

Stem cell culture apparatus further comprises a light source temperature control means for adjusting the temperature of the light source.
The method of claim 10,

The light source temperature control means stem cell culture apparatus characterized in that it comprises a cooling fan.
The method of claim 10, wherein the light source temperature adjusting means,

A refrigerant tank coupled to the light source;

An inlet hose whose one end is connected to the coolant tank to introduce a coolant;

An outlet hose whose one end is connected to the coolant tank to discharge the coolant; And

Stem cell culture apparatus comprising a refrigerant circulation portion connected to the other end of the inlet hose and the outlet hose to cool and circulate the refrigerant.
The method of claim 1,

Stem cell culture apparatus further comprises a distance adjusting means for adjusting the height of the light source up and down to adjust the distance between the container and the light source.
The method of claim 1,

The culture chamber further comprises a light-transparent transparent partition on the top and the light source is a stem cell culture apparatus for irradiating light into the culture chamber through the transparent partition.
The method of claim 14,

Stem cell culture apparatus further comprising a light blocking member that is a cover for blocking external light to the light source and the transparent partition.
The method of claim 14,

The light blocking member further comprises an air circulation means for circulating the air inside the sealing means with the outside.