Serum replacement composition and culture method using same

Abstract

The present invention provides: a serum replacement composition for cell culture, comprising an extract of Chlorella protothecoides; and a method for culturing cells, such as muscle stem cells, using the composition.

Description

Serum replacement composition and culture method using the same

[0001] The present invention relates to a serum substitute composition for cell culture comprising a Chlorella protothecoides extract; and a method for culturing cells such as muscle stem cells using the same.

[0002] Cultured meat is lean meat produced using cell engineering technology that cultivates meat without livestock farms by harvesting and culturing stem cells from living animals. In addition to the potential mitigation of climate change, cultured meat has the potential to address the environmental impacts of meat production, animal welfare, food security, and human health.

[0003] The culture of animal cells, including cultured meat, involves the use of media containing serum for cell growth and proliferation. Serums used for animal cell culture include fetal bovine serum (FBS), bovine calf serum (BCS), and horse serum (HS), with fetal bovine serum (FBS) being the most commonly used. Conventional animal cell culture media contain fetal bovine serum (FBS) at a concentration of approximately 10 v / v%. However, fetal bovine serums such as FBS present problems not only regarding ethical issues in the manufacturing process but also due to poor batch-to-batch uniformity caused by individual fetal variations and susceptibility to contamination. In particular, since the edible potential of cultured meat is not yet established and it is expensive, various studies are being conducted to develop different serum replacements in the cultured meat industry.

[0004] For example, Korean Patent Publication No. 10-2022-0138345 discloses the use of Chlorella extract as an additive to cell culture media. Korean Patent Publication No. 10-2022-0138345 discloses the results of evaluating serum replacement activity for an extract obtained from Chlorella vulgaris by a hot water extraction method. According to Korean Patent Publication No. 10-2022-0138345, for example in the culture of bovine muscle stem cells, when a hot water extract of Chlorella vulgaris is used at a concentration of 4 g / L, it exhibits approximately 80% FBS replacement activity. That is, the cell viability of bovine muscle stem cells cultured in the presence of a hot water extract of Chlorella vulgaris at a concentration of 4 g / L and 2% FBS is similar to the cell viability of bovine muscle stem cells cultured in the presence of 10% FBS. However, according to FIGS. 1 to 4 of Korean Patent Publication No. 10-2022-0138345, when the concentration of the hot water extract of Chlorella vulgaris exceeds 4 g / L, the cell viability decreases rapidly in a concentration-dependent manner, and thus the Emax of the hot water extract of Chlorella vulgaris is about 4 g / L, and the FBS replacement rate obtained at this time is about 80%.

[0005] Therefore, there is a demand in the industry for the development of serum substitutes that can effectively replace serums such as fetal bovine serum.

[0006] The inventors conducted various studies to develop serum replacements that can replace serums, such as fetal bovine serum, used for the growth and proliferation of animal cells. Surprisingly, the inventors discovered that a water-soluble extract obtained from the cells of a specific Chlorella strain [Chlorella protothecoides] using a non-hot water extraction process can be used as an effective serum replacement. In particular, the inventors discovered that when using the water-soluble extract, it exhibits an FBS replacement rate of up to 95% or more.

[0007] Accordingly, the present invention aims to provide a serum substitute composition for cell culture comprising a Chlorella protothecoides extract.

[0008] In addition, the present invention aims to provide a culture method comprising culturing cells in a medium containing the serum replacement composition.

[0009] According to one aspect of the present invention, a serum replacement composition for cell culture comprising a Chlorella protothecoides extract is provided.

[0010] According to another aspect of the present invention, a culture method is provided comprising culturing cells in a medium containing the serum replacement composition.

[0011] The present invention has revealed that a water-soluble extract obtained from the cells of a specific Chlorella strain, namely Chlorella protothecoides, using a non-hot water extraction process can be used as an effective serum substitute. In particular, the present invention has revealed that using the water-soluble extract can replace serum, such as FBS, by more than 95%. Therefore, the serum substitute composition according to the present invention and the cell culture method using the same can not only replace serum at a low cost but also be usefully employed in the production of cultured meat, etc., as they use edible materials.

[0012] The present invention provides a serum replacement composition for cell culture comprising a Chlorella protothecoides extract.

[0013] In the serum substitute composition of the present invention, the extract may be obtained by a manufacturing method comprising: (a) a step of obtaining cells from a culture medium of Chlorella protothecoides; and (b) a step of obtaining a water-soluble extract from the cells obtained in step (a) using a non-hot water extraction process.

[0014] The above Chlorella protothecoides may be a known Chlorella protothecoides strain, for example, Chlorella protothecoides DS-NCRC7 (KCTC 18633P) developed by the applicant may be used (Korean Patent Registration No. 10-2026681). The Chlorella protothecoides strain may be cultured according to the method disclosed, for example, in Korean Patent Registration No. 10-2026681. The culture medium of Chlorella protothecoides may be obtained in the form of a pellet by conventional methods such as centrifugation.

[0015] The above-mentioned water-soluble extract can be obtained from the cells obtained in step (a) using a non-hot water extraction process. For example, the above-mentioned non-hot water extraction process can be performed by adding water to the cells obtained in step (a), homogenizing them, and then obtaining a water-soluble extract. The water (e.g., purified water) can be added at a ratio of 3 to 10 ml, preferably about 5 ml, per 1 g of the cells obtained in step (a). The homogenization can be performed using a high-speed homogenizer at 10,000 to 18,000 rpm for 1 minute to 1 hour, preferably at about 14,000 rpm for about 30 minutes. The water-soluble extract can be obtained by separating the solid component and the supernatant from the homogenized material using conventional methods such as centrifugation. If necessary, the obtained water-soluble extract may be further filtered through a sterile filter (e.g., a 0.2 μm membrane filter) to obtain an extract in the form of an aqueous solution.

[0016] The obtained extract in the form of an aqueous solution can be used as is or concentrated as a serum substitute composition in the form of an aqueous solution containing water as a carrier. If necessary, the obtained extract in the form of an aqueous solution can be dried by conventional methods such as freeze-drying and used as a serum substitute composition in the form of a dried material that does not contain a carrier.

[0017] The present invention also provides a culture method comprising culturing cells in a medium containing the serum replacement composition.

[0018] In the culture method of the present invention, the cell may be an animal cell, for example, a muscle stem cell or a muscle cell. Preferably, the cell may be a bovine muscle stem cell or a porcine muscle stem cell.

[0019] In one embodiment, the present invention provides a method for culturing cells by replacing 80% or more of serum, such as FBS, which is typically used at a concentration of 10 v / v%. For example, in the culture method according to the present invention, the Chlorella protothecoides extract included in the serum replacement composition is present in the medium at a concentration of 0.1 g / L or more, preferably 0.13 to 0.23 g / L, and the serum may be present in the medium at a concentration of 2 v / v% or less.

[0020] In another embodiment, the present invention provides a method for culturing cells by replacing 90% or more of serum, such as FBS, which is typically used at a concentration of 10 v / v%. For example, in the culture method according to the present invention, the Chlorella protothecoides extract included in the serum replacement composition may be present in the medium at a concentration of 0.26 g / L or higher, and the serum may be present in the medium at a concentration of 1 v / v% or lower.

[0021] In another embodiment, the present invention provides a method for culturing cells by replacing 95% or more of serum, such as FBS, which is typically used at a concentration of 10 v / v%. For example, in the culture method according to the present invention, the Chlorella protothecoides extract included in the serum replacement composition may be present in the medium at a concentration of 0.29 g / L or higher, and the serum may be present in the medium at a concentration of 0.5 v / v% or lower.

[0022] The present invention will be explained in detail below through examples. However, the following examples are intended to illustrate the present invention, and the present invention is not limited by the following examples.

[0023] Example 1: Preparation of Chlorella protothecoides extract

[0024] Chlorella protothecoides DS-NCRC7 (KCTC 18633P) was fed-batch cultured in a 5 L jar fermenter for approximately 120 hours according to the method disclosed in Korean Patent Registration No. 10-2026681. The obtained culture medium was centrifuged to collect a cell pellet. 10 g of the obtained cell pellet was added to 50 ml of purified water and homogenized using a high-speed homogenizer at 14,000 rpm for 1 minute, followed by centrifugation at 3,000 rpm for 1 hour to separate the solid components from the supernatant. The supernatant was taken and filtered through a 0.2 µm filter to prepare an aqueous solution containing Chlorella protothecoides extract.

[0025] Example 2: Evaluation of serum replacement activity of Chlorella protothecoides extract in the proliferation of bovine muscle stem cells (bMuSC)

[0026] In the proliferation of bovine muscle stem cells (bMuSC), the serum replacement activity of the Chlorella protothecoides extract obtained in Example 1 was evaluated. Specifically, the effect on the proliferation of bovine muscle stem cells (bMuSC) following the addition of Chlorella protothecoides extract under culture conditions with reduced FBS was measured using the WST-8 test.

[0027] Specifically, bMuSCs with 3 subcultures were seeded at a rate of 5,000 per well in 96-well culture plates (3 wells per condition). The cells were cultured for one day in DMEM / F12 (Dulbecco's Modified Eagle's Medium / Nutrient Mixture F-12) containing 10 v / v% FBS at 37℃ and 5% CO2, and then half of the culture medium was replaced with DMEM / F12 containing different concentrations of FBS (0%, 0.5%, 1%, 2%, 10%) and Chlorella protothecoides extract (0 g / L, 0.03 g / L, 0.07 g / L, 0.1 g / L, 0.13 g / L, 0.16 g / L, 0.2 g / L, 0.23 g / L, 0.26 g / L, 0.29 g / L). The cells were allowed to adapt to the new culture medium by culturing for one day. The next day, the DMEM / F12 was completely replaced with one containing different concentrations of FBS (0%, 0.5%, 1%, 2%, 10%) and Chlorella protothecoides extract (0 g / L, 0.03 g / L, 0.07 g / L, 0.1 g / L, 0.13 g / L, 0.16 g / L, 0.2 g / L, 0.23 g / L, 0.26 g / L, 0.29 g / L), and cell viability was confirmed by performing WST-8 tests on days 0 and 6 starting from this point. The OD450 value measured on day 6 was determined by comparing it with the OD450 value measured on day 0.

[0028] As described above, the results of evaluating the serum replacement activity of Chlorella protothecoides extract in the proliferation of bMuSC are as shown in Table 1 below.

[0029] [Table 1]

[0030]

[0031] As can be seen from the results in Table 1 above, based on 6 days of bMuSC cell culture, the cell proliferation rate increased compared to the 10% FBS control group when Chlorella protothecoides extract was treated at concentrations of 0.1, 0.13, 0.16, 0.2, 0.23, 0.26, and 0.29 g / L under 2% FBS conditions (p>0.05). Under 1% FBS conditions, the cell proliferation rate increased compared to the 10% FBS control group when Chlorella protothecoides extract was treated at concentrations of 0.26 and 0.29 g / L. Under 0.5% FBS conditions, when Chlorella protothecoides extract was treated at a concentration of 0.29 g / L, there was no significant difference in cell proliferation rate compared to the 10% FBS control group (p>0.05).

[0032] Therefore, it can be seen that Chlorella protothecoides extract can replace FBS and induce cell proliferation of bMuSC. Specifically, it can be seen that when Chlorella extract is included in the total culture medium at a concentration of 0.1 g / L or more and less than 0.26 g / L, it shows an FBS replacement rate of 80% or more; when included at a concentration of 0.26 g / L or more and less than 0.29 g / L, it shows an FBS replacement rate of 90% or more; and when included at a concentration of 0.29 g / L, it shows an FBS replacement rate of 95% or more.

Claims

1. A serum replacement composition for cell culture comprising Chlorella protothecoides extract.

2. A serum substitute composition according to claim 1, characterized in that the extract is obtained by a manufacturing method comprising: (a) a step of obtaining cells from a culture medium of Chlorella protothecoides; and (b) a step of obtaining a water-soluble extract from the cells obtained in step (a) using a non-hot water extraction process.

3. A serum substitute composition according to claim 2, characterized in that Chlorella protothecoides is Chlorella protothecoides DS-NCRC7 (KCTC 18633P).

4. A serum substitute composition according to claim 2, characterized in that the non-hot water extraction process comprises adding water to the cells obtained in step (a) to homogenize them, and then obtaining a water-soluble extract.

5. A serum substitute composition according to claim 4, characterized in that water is added at a ratio of 3 to 10 ml per 1 g of bacterial cells obtained in step (a).

6. A serum substitute composition according to claim 2, characterized in that the manufacturing method further includes the step of filtering the water-soluble extract obtained in step (b) through a sterilization filter to obtain an extract in the form of an aqueous solution.

7. A culture method comprising culturing cells in a medium containing a serum substitute composition according to any one of claims 1 to 6.

8. A culture method according to claim 7, characterized in that the cell is a muscle stem cell or a muscle cell.

9. A culture method according to claim 8, characterized in that the cell is a bovine muscle stem cell or a porcine muscle stem cell.

10. A culture method according to claim 7, characterized in that the Chlorella protothecoides extract included in the serum substitute composition is present in the medium at a concentration of 0.1 g / L or higher, and the serum is present in the medium at a concentration of 2 v / v% or lower.

11. A culture method according to claim 10, characterized in that the Chlorella protothecoides extract included in the serum substitute composition is present in the medium at a concentration of 0.13 to 0.23 g / L.

12. A culture method according to claim 7, characterized in that the Chlorella protothecoides extract included in the serum substitute composition is present in the medium at a concentration of 0.26 g / L or higher, and the serum is present in the medium at a concentration of 1 v / v% or lower.

13. A culture method according to claim 7, characterized in that the Chlorella protothecoides extract included in the serum substitute composition is present in the medium at a concentration of 0.29 g / L or higher, and the serum is present in the medium at a concentration of 0.5 v / v% or lower.

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