KR101881281B1 - A Stem Cell Active Constituent Extracting Apparatus and A Stem Cell Active Extracting Method Using that - Google Patents

Abstract

The present invention relates to a stem cell active ingredient extracting apparatus and an extracting method using the stem cell extracting apparatus. The extracting apparatus includes a body portion in which a space is formed inside and an interior thereof is blocked, A second container part formed in the first container part and having a relatively small size relative to the first container part and having an opening at the top and into which stem cells are introduced; 2 a disruption unit for disrupting stem cells in the container unit, a valve for interrupting the flow of air into the body unit, and a pump for sucking air in the body unit.

Description

TECHNICAL FIELD [0001] The present invention relates to a stem cell extracting apparatus and an extracting method using the stem cell extracting apparatus.

TECHNICAL FIELD The present invention relates to a stem cell active ingredient extracting apparatus and an extracting method using the same, and more particularly, to a stem cell active ingredient extracting apparatus capable of increasing the collection rate of effective factors in stem cells and an extracting method using the same.

Stem cells are undifferentiated cells capable of differentiating into various types of body tissues. Many stem cell researches have been carried out because of their ability to differentiate into various tissue cells. Adult stem cells can be easily obtained from sites such as fat, bone marrow, remnant blood or placenta, have fewer ethical problems than embryonic stem cells, and use immune cells because they use their own cells. .

Implantation of adult stem cells is limited to the person's own reasons, such as immune rejection. So, instead of injecting stem cells, the culture medium obtained when the stem cells are cultured may be used. The culture medium by stem cell culture contains stem cells effective substances such as growth factors and cell active substances.

However, in addition to growth factors and cell active materials, such a culture medium contains other additives necessary for culturing such as bovine serum and antibiotics, and when such components are injected, they may have an undesired negative effect other than desired function or efficacy.

In addition, the culture medium in which the stem cells are cultured has a problem that the effective content of the stem cell is too low to obtain a desired effect, and it takes much time and effort to obtain a sufficient content.

Published Japanese Patent Application No. 10-2014-154754

An object of the present invention is to provide a method for producing a stem cell component extract capable of effectively extracting an effective ingredient of a stem cell.

In order to accomplish the above object, the present invention provides a stem cell active ingredient extracting apparatus comprising: a body part having an interior space where an interior space is formed and an interior thereof being blocked; a first container unit provided inside the body part, A second container portion formed in a relatively small size with respect to the first container portion and provided in the first container portion and opened upwardly and into which stem cells are injected; A crushing unit for crushing the stem cells, a valve for interrupting the flow of air into the body, and a pump for sucking air in the body.

And ice is loaded in the first container portion.

In another aspect of the present invention, there is provided a method for extracting a stem cell active ingredient, comprising the steps of: loading ice into a first container portion of the extraction device of Claim 1 and injecting stem cells into the second container portion; A third step of disrupting the stem cells by low-frequency ultrasonic waves; a fourth step of centrifugally separating the pulverized products of the third step; And a fifth step of filtering the separated extract with a triple filter.

According to the above-described constitution of the present invention, an effective ingredient of stem cells can be effectively extracted.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a drawing showing an apparatus for extracting an effective component of a stem cell according to the present invention. FIG.
FIG. 2 is a view showing a filter structure for extracting a stem cell active ingredient according to the present invention. FIG.

Hereinafter, preferred embodiments of the stem cell active ingredient extracting apparatus according to the present invention will be described in detail with reference to the accompanying drawings.

First, as shown in FIG. 1, the stem cell active ingredient extracting apparatus of the present invention comprises:

A first container portion 12 provided inside the body portion 10 and opening upward and a second container portion 12 provided inside the first container portion 12, A second container portion 14 formed in the first container portion 12 and opening upward to introduce stem cells into the inside of the second container portion 14, A valve 18 for interrupting the flow of air into the body 10 and a pump 20 for sucking air in the body 10, And the like.

First, the extracting apparatus of the present invention is provided with a body 10. As shown in FIG. 1, the body 10 is provided with an airtight space therein to block the outside and the inside thereof.

In the body 10, a first container portion 12 is provided. The first container portion 12 is formed to have a space therein and to open the upper portion thereof. Ice is loaded in the first container part 12 to lower the temperature of the stem cells inside the second container part 14 to be described below.

A second container portion (14) is provided in the first container portion (12). The second container portion 14 is formed such that a space is provided therein and an upper portion thereof is opened in the same manner as the first container portion 12. Particularly, the second container portion 14 is formed to have a size smaller than that of the first container portion 12, and the second container portion 14 can be loaded in the first container portion 12 have. Stem cells can be loaded in the second container part 14. [

Further, the crushing unit 16 may be further provided in the present invention. The crushing unit 16 serves to crush the stem cells loaded in the second container unit 14 by using ultrasonic waves.

In addition, the present invention is provided with a passage through which air flows from the outside, and a valve 18 for selectively blocking the flow of air through the passage is provided. At the other end of the flow path, a pump 20 is provided to discharge the air inside the body portion 10 to the outside so that the inside of the body portion 10 can be evacuated.

Hereinafter, a method for extracting an effective ingredient from a stem cell using the stem cell active ingredient extracting apparatus according to the present invention will be described.

First, the prepared stem cells are cultured in a medium. Here, stem cells can be extracted from any of human fat, bone marrow, remnant blood or placenta. The stem cells to be extracted are preferably adult stem cells, more preferably adult stem cells are mesenchymal stem cells.

The medium may be at least one of DMEM (Dulbecco's Modified Eagle Medium) and Keratinocyte-SFM (Keratinocyte serum free medium) which are widely used for stem cell culture. In addition, at least one of FBS (Fetal Bovine Serum), human serum, penicillin and streptomycin may be added to the medium, and nutrients such as vitamins, amino acids, lipids or growth factors may be further added. Preferably, FBS or human serum is added in an amount of 5 to 20%, penicillin or streptomycin is added in an amount of 0.1 to 5%, more preferably 8 to 15% in FBS or human serum, 2%.

The culture environment of the culture medium is preferably set at a humidity of 70 to 95%, a temperature of 20 to 50 DEG C and a concentration of 1 to 20% CO2. In some cases, such an environment can be arbitrarily changed.

In the step of culturing the stem cells, subculture can be performed. If the stem cells grow on a plate containing the medium and have a density of 80 to 90%, some stem cells are obtained from the plate and transferred to a plate containing another medium to allow the stem cells to grow again. If the blood is not removed during the primary culture, the blood can be removed while subculturing.

The medium is removed to obtain stem cells. The culture of the stem cells can be transferred to a large plate every time the subculturing step is performed, and when the subculture is carried out on a plate having a diameter of 10 cm or more, the medium is preferably removed to obtain stem cells. The above-mentioned stem cells can be obtained even on a plate smaller than a 10 cm plate, but a large amount of stem cells can not be obtained because the amount of stem cells cultured on a small plate is small. Therefore, obtaining stem cells from a plate of 10 cm or more is one of the ways to increase the working efficiency.

After removing the medium in the plate, trypsin / EDTA is added to separate. When trypsin / EDTA is added to stem cells, stem cells are stuck to the plate. The stem cells fallen from the plate are dried in a long bar shape to a round shape. Since trypsin / EDTA not only breaks the binding of the protein but also causes necrosis of the cells, it is added to the plate. After removing the trypsin / EDTA from the plate with 2 ~ 3 drops, the stem cells are obtained. The concentration of trypsin / EDTA is preferably 0.1 to 0.3%. It can also be scraped directly instead of trypsin / EDTA to recover. Plate-separated stem cells are washed several times with physiological saline or PBS (phosphate buffer saline) to completely remove trypsin / EDTA.

Stem cells are dispersed in an isotonic carrier liquid to count the number of stem cells while maintaining osmotic pressure. Isotonic carrier liquids generally use buffers such as physiological saline and phosphate buffered saline (PBS). In addition, they contain nutrients such as hartmann solution and hartmann glucose The carrier liquid can be used.

After the cultured stem cells are separated from the plate, an isotonic carrier liquid is added to the stem cells so that the cells can be disrupted, and then the stem cells are broken when the stem cells are flown so that they can be disrupted.

After the isotonic carrier liquid is removed, a storability enhancer is added to disperse the stem cells. The storability enhancer does not use an isotonic solution like carrier liquid but uses a solution with lower osmotic pressure than stem cells such as water and distilled water. When they are used, the stem cells expand quickly. If stem cells are to be left untreated for long periods of time in accordance with the process conditions for processing the stem cells, a saline solution having a higher salinity than water or distilled water, such as saline of 0.45% or less, may be used.

Since the storability enhancer has a lower osmotic pressure than the stem cells, when the expander is added to the stem cells, the stem cells absorb the expanding agent and become swollen and the cell membrane becomes thinner. It is preferable that the expanding agent is allowed to stand for 30 seconds to 2 minutes depending on the type thereof to sufficiently expand the stem cells. The extender preferably contains 10 ⁴ to 10 ⁷ stem cells per ml.

When the destruction energy such as ultrasonic waves is applied to the stem cells, the stem cells are easily broken because the cell membrane has already expanded sufficiently. Therefore, it is possible to effectively break down the cell membrane in a short time with a small energy, and it is possible to prevent the effective ingredient such as the growth factor inside the cell from being destroyed. In this case, the intensity of a suitable ultrasonic wave can be appropriately obtained even with ultrasonic wave which is remarkably lower than that of a normal cell disruption.

The disruption process of the stem cells is performed intermittently with a plurality of unit disruptions intervening the rest time. Stem cells can be damaged by the heat of shredding when the stem cells are continuously shattered without interrupting the downtime. The resting time allows the stem cells and extender to cool down and can be confirmed by microscopy to see if the breakdown is complete. After confirming that all of the stem cells have been disrupted through the microscope, the disruption is terminated. Excessive crushing may destroy not only cell membranes but also growth factors and cell active materials. It is preferable that each unit fracture is performed for 2 to 7 seconds in consideration of fracture efficiency and prevention of overheating, and the pause time is preferably 3 seconds or more.

In particular, when the stem cell active ingredient extracting apparatus of the present invention is used, low-frequency ultrasonic waves can be used in a low-temperature vacuum state, so that the ultrasonic wave can be broken even at a strength of 5 kHz or less, .

Finally, the stem cell component extract is finally isolated from the crushing step. In the mixture of the storability extender and the stem cell obtained in the above step, there are cell membrane residues together with various growth factors and cell active materials which are stem cell contents. Among them, growth factors and cell active materials necessary for the stem cell component extract are obtained, and unnecessary cell membrane residues are removed. In order to prevent the immune rejection reaction that may be caused by immunoglobulin, it is preferable to remove the destroyed cell membrane.

Removal of cell membrane residues can be done by filtering but mainly using centrifugation. The centrifugation is capable of recovering only the upper growth factors and cell active materials by allowing the cell membrane residues, which are larger than the growth factors and the cell active materials, to settle down.

The centrifugation is preferably performed at 800 to 1500G. When the concentration of the cell membrane is lower than 800 G, the cell membrane residue does not sink and the separation is not easy. When the concentration is higher than 1500 G, the amounts of the cell membrane residues as well as the growth factors and the cell active materials are reduced.

At this time, it is possible to filter out the stem cell disruption mixture by using the filter portion equipped with the filters 32, 34, and 46 capable of passing different molecular weights as shown in FIG. 2 during centrifugation.

Particularly, by repeating the filtering process by centrifugation several times, it is possible to extract the effective factors remaining in the cell wall and the like as much as possible.

Then, as shown in the following Table 1, the total amount of the protein was higher than that of the existing stem cell extract (when the stem cell extracting apparatus of the present invention was not used and the centrifuging filter of the present invention was not used) The result is 1.5 times more than the conventional method, and it can be confirmed that the more effective substances are extracted.

One 2 3 4 5 6 Average Previous method 58.28 56.29 55.97 56.05 56 55.73 56.39 The method of the present invention 96.97 95.46 96.83 96.01 96.46 96.94 96.45

(Unit: 占 퐂 / ml)

As described above, it is to be understood that the technical structure of the present invention can be embodied in other specific forms without departing from the spirit and essential characteristics of the present invention.

Therefore, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than the foregoing description, All changes or modifications that come within the scope of the equivalent concept are to be construed as being included within the scope of the present invention.

10: body part 12: first container part
14: second container part 16: crushing part
18: valve 20: pump
30:

Claims (3)

delete delete A body portion having a space formed therein and intercepting the inside and outside of the body portion;
A first container portion provided inside the body portion and opening upward, and in which ice is loaded;
A second container part formed in a relatively small size with respect to the first container part and provided inside the first container part and opened upward so as to inject stem cells therein;
A crushing unit for crushing the stem cells inside the second container unit by ultrasonic waves;
A valve for interrupting the flow of air into the body portion; And
And a pump for sucking air in the body part,
Culturing the stem cells in a culture medium, washing the stem cells after removing the culture medium, counting the number of stem cells by dispersing the stem cells in an isotonic carrier liquid, removing the carrier liquid, The stem cell is added to the stem cell to disperse the stem cell in an amount of 10 ⁴ to 10 ⁷ per 1 ml of the extender, the ice is loaded into the first container, and the stem cell is introduced into the second container ;
A second step of driving the pump to evacuate the inside of the body part;
A third step of driving the crushing unit to crush the stem cells by low-frequency ultrasonic waves;
A fourth step of centrifugally separating the crushed material in the third step;
And a fifth step of filtering the centrifuged extract with three filters capable of passing different molecular weights,
The hypothesis of the third stage has a duration of 2 to 7 seconds at an intensity of 5 kHz and a dwell time of 3 seconds,
Wherein the centrifugal separation in the fourth step is performed at 800 to 1500G.

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