KR20210086811A - A effective method for culturing cell - Google Patents

Abstract

The present invention relates to a cell culture method suitable for mass production. In the present invention, tissues can be minced automatically using a grinder, and thus cells can be efficiently cultured. The method comprises the following steps of: collecting a muscle sample from a pig; mincing the collected muscle sample; culturing the minced muscle sample; and isolating muscle satellite cells.

Description

An effective cell culture method {A EFFECTIVE METHOD FOR CULTURING CELL}

The present invention relates to an effective cell culture method. According to one embodiment of the present invention, it is possible to effectively culture porcine muscle satellite cells and preadipocytes.

[Pig muscle satellite cells]

A muscle satellite cell is a cell located on the outer surface of a muscle fiber, and exists between the sarcolemma and basal lamina, which are the edges of the muscle. When damaged or destroyed, they are activated and differentiated and are adult stem cells that elongate muscles.

Muscle satellite cells were discovered in 1961 by Alex Mauro. After that, in 2008, researchers at Pompeu Fabra University announced the process of muscle building from muscle satellite cells, and muscle satellite cells are being actively used as the subject of various studies.

[Conventional cell culture method]

Cell culture is the most basic research method in bio-field research and is widely used in research on the function of living things.

Cell culture proceeds comprising collecting a sample from a subject, culturing the cells in a suitable culture medium for the collected sample, and isolating the obtained cells.

For example, the following method is known as a muscle satellite cell culture method.

The muscle tissue is isolated from the muscle using the Dodson method. The isolated muscle is washed with PBS buffer, and external connective tissue and blood vessels are removed on a clean bench. The muscle is cut into ^{1-2 cm 3 segments.} The cut muscle is treated with a pronase solution (0.8 mg/ml pronase, type XIV, in PBS) in a 37 °C water bath and incubated for 1 hour with stirring.

After incubation, the tissue mixture is centrifuged at 1,500 g for 6 minutes and the supernatant is aspirated. The cell precipitate is washed once with PBS, and after centrifugation under the above conditions, the final precipitate is resuspended in PBS.

Muscle satellite cells were isolated from the myotubes by washing 3 times for 10 min at 400 g. Combine all of the supernatant containing muscle satellite cells and centrifuge at 1,800 g for 10 min. The cell precipitate is suspended in DMEM (Dulbecco's modification of Eagle's MEM) + 10% HS (horse serum) culture medium, and filtered through 500um Nitex cloth (Tetko Inc., Elmsford, NY).

The cell suspension was pre-plated in a cell culture vessel with a diameter of 15 cm, and _{incubated for 2 hours at 37° C. in a humidified CO 2} incubator (Forma scientific, USA) (5% CO ₂ + 95% air).

The effect of pre-plating can be to remove contaminants in the float. After pre-culture, the suspension was centrifuged at 1,500 g for 10 minutes, and the cell precipitate was re-suspended in DMEM + 10% HS. The cell suspension was filtered through a 53um Nitex cloth, and the muscle satellite cell suspension was plated in a cell culture vessel and incubated in DMEM + 10%HS.

However, the above conventional method is not productive in culturing cells in a large amount of industrial capacity. The reason is that when cutting to a certain size for culturing the isolated muscle, the operator must individually and manually cut it.

For example, even in the example of Korean Patent Application Laid-Open No. 10-2019-0141137, after collecting horse skeletal muscle, the specimen is handled very carefully to prevent damage to the muscle tissue, and with care in PBS, the length of one side is about 1 mm The process of incision is shown in very small slices.

Even in the primary culture method listed in the textbook, the tissue pieces are ^{manually cut into small pieces of 1 to 2 mm 2 and} then cultured. This is because, if the tissue is cut too large, the growth rate of the cells may be very slow, but if the cells are cut randomly with a blender rather than manually, the delicate plasma membrane may be destroyed and cells may be damaged.

Republic of Korea Patent Publication No. 10-2019-0141137

Pig muscle satellite cells are required to be industrially mass-produced for research. However, in the conventional method, the process of individually and delicately shredding the sample by the operator is essential, and there is a limit to mass production according to automation.

The present inventors have come to complete the present invention in order to improve the limitations of manual operation in cell culture.

The present invention has solved the above-mentioned problems by means of the following means.

1. (1) collecting a muscle sample from a pig, (2) mincing the collected muscle sample, (3) culturing the minced muscle sample, and (4) isolating the muscle satellite cells. A method for culturing muscle satellite cells of pigs, comprising: automatically slicing each side to a size of 0.5 - 0.7 cm using a grinder in step (2).

2. The method for culturing porcine muscle satellite cells according to the above 1, characterized in that in step (2), an antibiotic at 4° C. is additionally supplied to the grinder and minced.

3. The method for culturing porcine muscle satellite cells according to 1 or 2 above, characterized in that the pulverization process is repeated a total of two times in step (2) and minced.

4. The method according to any one of 1 to 3 above, further comprising removing connective tissue, capsule, blood vessels and fat from the muscle collected in step (1) before proceeding to step (2) and putting it in an antibiotic at 4°C A method for culturing muscle satellite cells in pigs, characterized in that

The present invention can reduce the total cell extraction time for cell culture from approximately 1 hour to 15 minutes compared to a case in which a conventional operator manually chops a sample.

The present invention has superior work efficiency compared to the case in which a conventional worker manually cuts a sample with scissors or the like, so that industrialization such as mass production is possible.

For reference, when cutting with scissors in the prior art, physical difficulties were also considerable for workers, such as straining their hands, but according to the present invention, the above limitations are also overcome.

1 is a diagram showing piglet euthanasia (left) and a schematic diagram of hind leg muscles (right).
2 is a view showing morphology according to the number of pulverization.
3 is a diagram showing cell morphology 24 hours after dispensing the manually minced (Comparative Example 1) cells.
Figure 4 is a diagram showing cell morphology 24 hours after dispensing the cells chopped (Example 1) with a grinder.
Figure 5 is a diagram comparing the number of cells in the case of using a manual (Comparative Example 1) and a grinder (Example 1).
Figure 6 is a view showing the blade spacing of the grinder of one embodiment according to the present invention. The unit of the ruler in the drawing is cm.

For muscle satellite cell extraction, two-week-old piglets were prepared and euthanized, and Biceps femoris muscle was isolated and extracted from the hind leg (FIG. 1).

Dulbecco's phosphate-buffered saline (hereinafter referred to as DPBS, WELGENE Inc., Gyeongsan) added with antibiotics (2X anti-biotics-antimycotic, AA, Gibco, Waltham, MA, USA) at 4℃ to prevent contamination. , Korea) solution and transported to a clean bench, and subsequent experiments were carried out under sterile conditions.

The connective tissue, capsule, blood vessel, fat, etc. were removed from the muscle tissue, and this was put in a grinder and grinded twice in total.

The grinder sets the blade spacing so that one side of the sliced tissue is 0.5 - 0.7 cm in size.

The grinder includes a blade and an automatically rotating screw. In one embodiment according to the present invention, the tissue may pass through 3 to 6 blades according to the automatically rotating screw, so that one side of the size of the chopped tissue may be shredded to have a size of 0.5 - 0.7 cm. At this time, the interval of each of the 3 to 6 blades may be arranged in a gradually narrowing manner, and the interval of the last blade may be arranged to be 0.5 - 0.7 cm (FIG. 6). In this way, it was confirmed that the number of cultured cells can be more effective as cell damage is minimized when the tissue is gradually reduced in size by sequentially arranging the blade gaps narrowly.

In addition, compared to the case of pulverizing once, pulverizing twice can ensure more uniformity in the size of the tissue. However, when pulverized 3 times, more uniformity can be ensured than when pulverized twice, so that the cell culture effect was expected to be excellent, but the number of cells after culture was rather low compared to the case of pulverization twice. In terms of cell culture effect and efficiency, 2 times less than 3 times may be more preferable.

Conventionally, it has been common for the operator to directly mince the cells for cell culture. The reason was that there was a risk of damage to the cells in the process of shredding the tissue. However, in the case of direct shredding, it may take a long time, and especially when slicing with scissors, the fatigue applied to the hand is considerable, and the work efficiency is not good. In addition, in the case of manual shredding by an operator, there was a limitation in that the number of cell cultures was not constant due to variation in the size of the tissue.

However, the present inventor surprisingly confirmed that sufficient cell culture is possible when the size of one side of the grinder is set so that the sample can be automatically minced to a size of 0.5 - 0.7 cm. However, in the case of pulverizing with a grinder, cell culture was not effective in all cases, and in the case of shredding smaller or larger than the above size, the exact principle is unknown, but a difference occurred in the number of cell cultures and it was not effective.

If the sample is shredded automatically through the grinder, the time required for cell extraction can be reduced from about 1 hour to about 15 minutes compared to the case of shredding by an operator, and it is effective because there is little deviation between each cell culture or each person in charge. . In particular, the present invention has a significant difference in effect compared to the point that the worker cuts in the point that it guarantees the possibility of mass production on an industrial scale.

When shredding in a grinder, it is preferable to additionally supply an antibiotic to the grinder if necessary. The cell culture effect could be better when the antibiotics at 4 ° C were supplied to the grinder and grinded.

Then, 10 g of the pulverized tissue was placed in a 50 mL tube, 20 mL of pronase (0.8 mg pronase/mL DPBS; Dulbecco's phosphate buffered saline+AA; antimycotic-antibiotic) solution was added, and incubated in a constant temperature bath at 37°C for 40 minutes .

After pronase treatment, the tissue was centrifuged at 1200 x g for 15 minutes, the supernatant was discarded, and the pellet was mixed with 20 mL of DPBS, and centrifuged again at 1200 x g for 15 minutes.

After that, the settled pellet was diluted in a solution of minimum essential medium (MEM, Gibco, Waltham, MA, USA) containing AA, centrifuged at 300 x g for 5 minutes, and the process of collecting the supernatant was repeated twice, and the supernatant was repeated twice. Centrifuge the solution again at 1200 x g for 15 minutes, mix the settled pellet with 9 mL of MEM+AA+10% FBS solution, filter it with a 100-μm cell strainer, and dispense 1 mL of the filtrate into cryovials. DMSO was added to make it 10%, put in a freezing container, and stored frozen at -70°C.

Frozen vials were thawed in a constant temperature water bath at 37°C, mixed with culture medium added with 10% FBS to MEM warmed, and centrifuged at 300 x g for 5 minutes. The pellets were mixed with the culture medium and dispensed into a culture flask.

The seeded cells were cultured in a 37° C. CO ₂ incubator, and the medium was replaced 24 hours after dispensing, and then the medium was replaced every 48 hours.

After the cells have grown beyond a certain level (over 80% confluent), the cells are washed with PBS pre-warmed in a 37°C water bath, treated with Trypsin-EDTA _{, incubated in a 37°C CO 2} incubator for 5 minutes, and the medium is added. The trypsin activity was stopped.

Thereafter, the supernatant was removed by centrifugation at 2500 rpm for 3 minutes, and the subcultured pellets were subcultured.

When freezing cells, the pellet after centrifugation is mixed with the medium, DMSO is added to make it 10%, dispensed into cryovials, put in a freezing container, and frozen in a deep freezer.

There are many products sold as freeze-drying agents for freeze-drying. However, there was no significant difference in cell yield and growth rate in the cells frozen with each cryoculture medium. Therefore, a simple and inexpensive freezing method using DMSO is effective in the present invention.

In addition, after freezing at 4°C for 12 hours and at -20°C for 4 hours, the method was stored at -70°C, and the cells were placed in a container filled with isopropanol and immediately frozen at -70°C (cell freezer). As a result, it was confirmed that putting it in a cell freezer container reduced the deviation and made it simpler.

In order to keep the state of the cells constant, the number of cells was counted and the number of cells to be dispensed on the plate was constant before subculture, and 10 mL of medium was mixed with the centrifuged cells to dissolve them well. After mixing 10 μL of medium and 10 μL of trypan blue solution, the cells were counted in a hematocytometer.

/well 의 세포를 분주하였다.Cells inactive by tryphan blue were not included in the count because they were stained blue when counting the cells, and the amount of cells to be dispensed was 6 well 2×10

/well cells were aliquoted.

Cell doubling time or population doubling time (PDT) is the average time required to double the growth rate of cells. The number of cells proliferated for 24 hours was counted for one week, a growth curve was drawn, and PDT was obtained using the following formula.

Differentiation was induced using differentiation medium (DMEM + 2% horse serum) in muscle satellite cells grown to be more than 90% confluent.

To observe the morphology of the cultured muscle cells, the nucleus of the cells was stained with Giemsa.

After washing the muscle cells with PBS, the cells were fixed with 100% MeOH, dried completely, and the monolayer of the fixed cells was treated with giemsa stain solution for 30 minutes, and then washed with distilled water so that the staining outside the cell nucleus was discolored. It was observed under a microscope.

The present invention will be described in more detail with reference to the following examples.

Below, the remaining experimental methods and conditions, including tissue resection, were applied in the same manner as the above-described process, and the number of cell cultures was compared by setting the sample shredding method differently. When shredding in a grinder, an antibiotic at 4°C was supplied to the grinder and minced.

In the case of manual shredding, the length of one side of the sample was measured using a ruler after shredding with scissors.

In the case of automatic shredding using a grinder, the representative size of the minced sample was measured through a sample test after setting the grinding conditions of the grinder by adjusting the distance between the blades of the grinder.

[Comparative Example 1]

The minced tissue was manually minced using scissors so that one side of it was about 1 mm.

[Comparative Example 2]

A grinder with an adjustable blade interval was prepared so that one side of the chopped tissue could be cut into pieces of 0.1 - 0.3 cm, and the slices were repeated twice.

[Comparative Example 3]

One side of minced tissue 1 - 1.5 cm A grinder with the interval of the blade adjusted so that it can be chopped to the size of

[Example 1]

A grinder with an adjustable blade interval as shown in FIG. 6 was prepared so that one side of the sliced tissue could be cut into pieces of 0.5 - 0.7 cm, and the slices were repeated twice.

[Example 2]

As shown in FIG. 6, a grinder with an adjustable blade interval was prepared so that one side of the minced tissue could be cut into pieces of 0.5 - 0.7 cm, and the cut was repeated three times.

[Example 3]

As shown in FIG. 6 , a grinder with an adjustable blade interval was prepared so that one side of the minced tissue could be cut to a size of 0.5 - 0.7 cm, and the shredded pieces were repeated once.

[Experimental Example 1] Comparison of the number of cells according to the number of grinding

The muscle cell yield was affected by the number of crushing of the muscle slices, and when the crushing was performed twice, the particle size did not differ significantly with the naked eye compared to the case of crushing once or crushing 3 times (FIG. 2).

However, the cell yield was highest in the sample pulverized twice compared to the case pulverized once or three times.

The result is as follows.

cell fluid Example 3 Example 1 Example 2 100 uL 5.00E+02 6.00E+03 5.00E+03 200 uL 7.00E+03 1.20E+04 1.00E+04 300 uL 9.00E+03 5.00E+04 4.00E+04 400 uL 2.40E+04 9.00E+04 7.50E+04 500 uL 8.33E+05 2.10E+06 1.00E+06 1000 uL 1.20E+06 5.20E+06 3.43E+06

[Experimental Example 2] Comparison of the number of cells by hand (mincing) and grinding (grinding)

As in the cell morphology of Figure 3, the cell yield in the tissue ^{minced by hand after 24 hours of Comparative Example 1 was 1X10 4} (100uL dispensed), 4X10 ⁴ (200uL dispensed), 7X10 ⁴ (300uL dispensed), 1.4X10 ⁵ ( 400uL dispensed), 2.5X10 ⁶ (500uL dispensed), and 5.9X10 ⁶ (1000uL dispensed).

The cell yield in the tissue minced with the grinder of Example 1 (FIG. 4) was 6X10 ³ (100uL dispensed), 1.2X10 ⁴ (200uL dispensed), 5X10 ⁴ (300uL dispensed), 9X10 ⁴ (400uL dispensed), 2.1X10 ⁶ (500uL dispensed), 5.2X10 ⁶ (1000uL dispensed).

cell fluid Comparative Example 1 Example 1 100 uL 1.00E+04 6.00E+03 200 uL 4.00E+04 1.20E+04 300 uL 7.00E+04 5.00E+04 400 uL 1.40E+05 9.00E+04 500 uL 2.53E+06 2.10E+06 1000 uL 5.90E+06 5.20E+06

As shown in the table above, 100, 200, 300, 400, 500, and 1000 uL of cell fluid extracted from the tissue minced with a hand or grinder was dispensed and the number of cells was compared after 24 hours. As a result, there was no statistical difference between the two groups (Fig. 5).

[Experimental Example 3] Comparison of the number of cells according to the grinding size

The cell numbers of Comparative Examples 2 and 3 and Example 1 were compared.

cell fluid Comparative Example 2 Comparative Example 3 Example 1 100 uL 4.50E+01 7.00E+01 6.00E+03 200 uL 2.00E+02 5.80E+02 1.20E+04 300 uL 5.00E+02 0.20E+03 5.00E+04 400 uL 9.70E+02 5.00E+03 9.00E+04 500 uL 3.33E+03 9.10E+03 2.10E+06 1000 uL 8.20E+03 1.40E+04 5.20E+06

In the case of automatic pulverization using a pulverizer, cell culture was not effective in all cases, and it was confirmed that a sufficient number of cells were cultured only in Example 1.

Claims (4)

(1) collecting a muscle sample from a pig, (2) mincing the collected muscle sample, (3) culturing the minced muscle sample, and (4) isolating the muscle satellite cells. A method for culturing porcine muscle satellite cells, characterized in that in step (2), using a grinder, each side is automatically minced to a size of 0.5 - 0.7 cm. The method according to claim 1, wherein in step (2), antibiotics at 4° C. are additionally supplied to the grinder and minced. [Claim 3] The method for culturing porcine muscle satellite cells according to claim 1, wherein the grinding process is repeated twice in step (2). The pig according to claim 1, further comprising the step of removing connective tissue, capsule, blood vessels and fat from the muscles collected in step (1) before proceeding to step (2) and putting them in antibiotics at 4 ° C. of muscle satellite cell culture method.

Images