CN114292809A - Culture medium containing chicken serum for in vitro cell culture and application of chicken blood in freshness - Google Patents
Culture medium containing chicken serum for in vitro cell culture and application of chicken blood in freshness Download PDFInfo
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- CN114292809A CN114292809A CN202111677495.8A CN202111677495A CN114292809A CN 114292809 A CN114292809 A CN 114292809A CN 202111677495 A CN202111677495 A CN 202111677495A CN 114292809 A CN114292809 A CN 114292809A
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Abstract
The invention belongs to the technical field of in vitro cell culture, and particularly relates to a culture medium for in vitro cell culture containing chicken serum and application of the chicken serum in fresh chicken blood.
Description
Technical Field
The invention belongs to the technical field of in vitro cell culture, and particularly relates to a culture medium containing chicken serum for in vitro cell culture and application of chicken serum in freshness.
Background
From the 50 s of the 20 th century, animal serum is widely applied to cell culture systems as an additive, and good culture effects are achieved, such as higher growth density of cells, higher activity and more cell passages, wherein mammalian serum (such as cow serum, sheep serum and horse serum) is more applied to primary cell culture, passage culture and cell line culture, but poultry serum is relatively less applied, especially chicken serum. The chicken is the most common poultry raised by human beings and is mainly used as food material, while the chicken blood rich in various active ingredients is abandoned as a byproduct, but the serum in the chicken blood contains various plasma proteins, polypeptides, hormones, lipids and other abundant cytokines for promoting cell growth, thereby being beneficial to cell proliferation and passage.
Disclosure of Invention
In order to solve the technical problems, the invention provides a culture medium containing chicken serum for in vitro cell culture and application of chicken serum for freshening, and the chicken serum is used as a culture medium additive to provide a new animal serum for in vitro cell culture.
The invention is realized by firstly providing a culture medium for in vitro cell culture containing chicken serum, and the components comprise the chicken serum and components required by conventional in vitro cell culture.
In addition, a new application of the chicken serum is provided, namely the chicken serum is used for preparing a culture medium for in vitro cell culture.
Compared with the prior art, the invention has the advantages that:
the method provides animal serum selection suitable for in vitro cell culture, namely chicken serum, which is generally suitable for in vitro amplification of primary cells, passage cells and cell lines and provides a new way for in vitro culture of the primary cells, the passage cells and the cell lines.
Drawings
FIG. 1 is a flow chart of chicken serum collection and cell culture;
FIG. 2 is a photograph of a primary cell (human umbilical cord mesenchymal stem cell) culture (FIG. 2-1 shows, in order from left to right, 2% of serum P1, P4 and P8 generation cells of Canada chicken, FIG. 2-2 shows, in order from left to right, 2% of serum P1, P4 and P8 generation cells of Canada chicken, FIG. 2-3 shows, in order from left to right, 2% of serum P1, P4 and P8 generation cells of Canada chicken, FIG. 2-4 shows, in order from left to right, 5% of serum P1, P4 and P8 generation cells of Canada chicken, FIG. 2-5 shows, in order from left to right, 5% of serum P1, P4 and P8 generation cells of Canada chicken, FIG. 2-6 shows, in order from left, 5% of serum P1, P4 and P8 generation cells, FIG. 2-7 shows, in order from left, 10% of serum P56, P4 and P8672 and P869 generation cells of Canada chicken, and FIG. 2-7 shows, in order from left, P4, P8 generation cells; 2-10 from left to right for 10% FBS P1, P4, P8 generation cells);
FIG. 3 is a photograph of a subculture cell (human umbilical vein endothelial cell) (FIG. 3-1 shows sequentially from left to right 2% of serum P1, P4 and P8 of chicken, FIG. 3-2 shows sequentially from left to right 2% of serum P1, P4 and P8 of chicken, FIG. 3-3 shows sequentially from left to right 2% of serum P1, P4 and P8 of shoal chicken, FIG. 3-4 shows sequentially from left to right 5% of serum P1, P4 and P8 of chicken, FIG. 3-5 shows sequentially from left to right 5% of serum P1, P4 and P8 of shoal chicken, FIG. 3-6 shows sequentially from left to right 5% of serum P1, P4 and P8 of shoal chicken, FIG. 3-7 shows sequentially from left to right 10% of serum P1, P4 and P8 of chicken, FIG. 3-8 shows sequentially from left 10% of serum P1, P4 and P8672 and FIG. 3-7 shows sequentially from left to right 10% of the shoal chicken serum P1, P4, P8 generation cells; 3-10 from left to right for 10% FBS P1, P4, P8 generation cells);
FIG. 4 is a photograph of a cell line (human dermal fibroblast) cultured (FIG. 4-1 shows, in order from left to right, 2% of serum P1, P4 and P8 generation cells of Canada chicken, FIG. 4-2 shows, in order from left to right, 2% of serum P1 of Bombarus domestica, P4 and P8 generation cells of Bombarus domestica, FIG. 4-3 shows, in order from left to right, 2% of serum P1, P4 and P8 generation cells of Bombarus domestica, FIG. 4-4 shows, in order from left to right, 5% of serum P1, P4 and P8 generation cells of Canada chicken, FIG. 4-5 shows, in order from left to right, 5% of serum P1, P4 and P8 generation cells of Bombarus domestica, FIG. 4-6 shows, in order from left to right, 5% of serum P1, P4 and P8 generation cells of Bombarus domestica, FIG. 4-7 shows, in order from left to right, 10% of serum P1, P4 and P8672 and P869 generation cells of Bombarus domestica, P4, P8 generation cells; FIGS. 4-10, from left to right, are 10% FBS P1, P4, P8 generation cells) in sequence.
Detailed Description
The present invention is further illustrated by the following specific embodiments, but is not intended to limit the scope of the present invention.
At present, cell culture and clinical application tend to serum-free culture, toxic effect and source pollution of animal serum to cells can be avoided, but the serum-free culture generally has stronger pertinence, and one serum-free culture medium is only suitable for culture of certain cells, so that the defects of higher use cost and the like are caused. Compared with mammals, the poultry and the human have far genetic relationship, less cell-derived pollution and research value in cell culture. The chicken is a representative animal of poultry, is used as a main source of meat of people, has the advantages of easy acquisition and low cost, and the chicken blood is usually treated as waste, thereby wasting resources and polluting the environment. Based on the culture medium, the invention provides a culture medium containing chicken serum for in vitro cell culture and application of chicken blood freshening.
Example of the implementation
Selecting three common chicken varieties in northeast China: forest meadow chicken, big bone chicken and shou light chicken are bred for 14-16 weeks under the same daily ration and breeding conditions, 3 healthy big cocks are selected for each chicken for venous blood collection, chicken serum is verified to be used as an additive, primary cells, passage cells and cell lines can be cultured in vitro, cell growth is promoted, the process is referred to as a figure 1, and the method specifically comprises the following steps:
process for preparing chicken blood serum
1. Chicken blood Collection
1) Blood collection: taking blood from lateral axillary vein of chicken by using the existing aseptic chicken blood collection operation rules;
2) collecting: transferring the chicken blood in the blood collecting tube into a 50mL centrifugal tube;
2. preparation of chicken serum
1) Centrifuging: 4000rpm, 20 ℃, 10 min; after centrifugation, taking the upper serum to a new 50mL centrifuge tube;
2) and (3) filtering: filtering the serum in the centrifugal tube for 3 times by using a 0.22 mu m filter;
3) subpackaging: and (4) subpackaging the filtered chicken serum.
Secondly, in vitro culture of primary cells, passage cells and cell lines (verification experiment):
1. primary cell culture (umbilical cord was chosen for its typicality):
1) tissue origin: provided by the Jilin national health Life Bank;
2) the culture method comprises the following steps: taking a normal healthy newborn umbilical cord which is born in full term by 10cm, ligating threads at two ends, and soaking in a DMEM-containing storage bottle. Taking out umbilical cord from super clean bench, sterilizing umbilical cord surface with 75% ethanol for 2-3min, cutting off ligature silk at two ends, washing residual blood with normal saline, cutting umbilical cord into 2cm segments, rinsing again, dissecting umbilical cord, removing umbilical vein and umbilical artery, and stripping Wharton's jelly. Shearing Wharton jelly into size of 1mm × 1mm × 1mm, inoculating into DMEM culture bottle containing 2%, 5% and 10% chicken serum, respectively, placing at 37 deg.C and 5% CO2Culturing in an incubator.
3) Positive control: in order to eliminate the influence of factors such as experimental operation and experimental environment on the cell growth state, a control group is set: DMEM with 10% FBS.
2. Subculture of cells
1) Cell source: primary human umbilical vein endothelial cells were purchased by ATCC Call Lines official website;
2) cell subculturing: according to the culture method of human umbilical vein endothelial cells provided by ATCC Call Lines official network, standard experimental operation, after counting cells, inoculating the cells into a DMEM six-hole plate containing 2%, 5% and 10% of chicken serum according to 4.8 × 104 cells/hole, respectively, placing the cells into a DMEM six-hole plate at 37 ℃ and 5% of CO2Culturing in an incubator.
3) Positive control: in order to eliminate the influence of factors such as experimental operation and experimental environment on the cell growth state, a control group is set: DMEM with 10% FBS.
3. Cell line culture
1) Cell source: primary human dermal fibroblasts were purchased by ATCC Call Lines official website;
2) cell culture: according to the culture method of human umbilical vein endothelial cells provided by ATCC Call Lines official network, standard experimental operation, after counting cells, inoculating the cells into a DMEM six-hole plate containing 2%, 5% and 10% of chicken serum according to 4.8 × 104 cells/hole, respectively, placing the cells into a DMEM six-hole plate at 37 ℃ and 5% of CO2Culturing in an incubator.
3) Positive control: in order to eliminate the influence of factors such as experimental operation and experimental environment on the cell growth state, a control group is set: DMEM with 10% FBS.
Thirdly, verifying the result
1. Primary cell culture
TABLE 1 Primary cell culture quantity results Table
From the above results and fig. 2, it can be seen that:
(1) the cytological morphology is as follows: observing the growth spindle and the mononuclear of the cells under a microscope, wherein the cell forms tend to be consistent after passage and are arranged in a radial or vortex shape, the cells can be stably passed, and the cell growth conditions are not too different in at least 8 generations, which is shown in figure 2 in detail;
(2) cell number: according to statistics of cell proliferation quantity, the cell quantity of DMEM containing 5% and 10% of chicken serum is stably proliferated, the cell is stably transferred to 8 th generation, and the proliferation quantity has no great difference; DMEM with 2% chicken serum, slightly less cell number; in conclusion, the primary judgment shows that the effect is best when the mesenchymal stem cells are cultured in DMEM with 5% -10% of chicken serum;
(3) flow cytometry detection: the results of flow detection of the mesenchymal stem cells cultured by DMEM with 2%, 5% and 10% chicken serum show that the phenotype of the mesenchymal stem cells is unchanged along with the increase of the cell passage frequency, and the phenotype of the mesenchymal stem cells is high in expression of CD73, CD90 and CD105, low in expression of CD14, CD19, CD34, CD45 and HLA-DR.
The biological characteristics of cell morphology, proliferation, phenotype and the like are well maintained after the cells are cultured until the generation P8, which indicates that the chicken serum can be used as an additive for the in vitro culture and the promotion of cell growth of primary cells in the cell culture.
2. Subculture of cells
TABLE 2 table of the number of subcultured cells
As can be seen from the above results and from fig. 3,
(1) the cytological morphology is as follows: the cells were observed under a microscope for single or colony-like growth. The single cell grows in a fusiform shape, an oval shape or a polygonal shape; the colony-shaped cells are densely arranged at the center and loosely arranged at the edge, and then the cells can be stably passaged by paving stones or whirlpool growth, and the cell growth conditions are not too different within at least 8 generations, which is detailed in figure 3;
(2) cell number: according to statistics of cell proliferation quantity, the cell quantity of DMEM containing 5% and 10% of chicken serum is stably proliferated, the cell is stably transferred to 8 th generation, and the proliferation quantity has no great difference; DMEM with 2% chicken serum, slightly less cell number; in conclusion, the primary judgment shows that the effect is best when the mesenchymal stem cells are cultured in DMEM with 5% -10% of chicken serum;
(3) flow cytometry detection: the phenotype of the high expression CD29, CD31, CD44, CD54, CD105, CD166, low expression CD34, CD49 and CD106 is not changed along with the increase of the cell passage number according to the flow detection result of the mesenchymal stem cells cultured by DMEM with 2%, 5% and 10% of chicken serum.
The biological characteristics of cell morphology, proliferation, phenotype and the like are well maintained after the culture of the P8 generation cells, which indicates that the chicken serum can be used as an additive for the in vitro culture of the subculture cells and the promotion of cell growth in the cell culture.
3. Cell line culture
TABLE 3 results of cell line culture number
From the above results and fig. 4, it can be seen that:
(1) the cytological morphology is as follows: the cells were observed under microscope to be larger in size, mainly fusiform, with oval nuclei, having morphological characteristics of fibroblasts, as detailed in fig. 4.
(2) Cell number: according to statistics of cell proliferation quantity, the cell quantity of DMEM containing 5% and 10% of chicken serum is stably proliferated, the cell is stably transferred to 8 th generation, and the proliferation quantity has no great difference; DMEM with 2% chicken serum, slightly less cell number; in conclusion, the primary judgment shows that the effect is best when the mesenchymal stem cells are cultured in DMEM with 5% -10% of chicken serum;
(3) flow cytometry detection: the high expression of CD29, CD73, CD30 and CD105 is realized through the flow detection result of the mesenchymal stem cells cultured by DMEM with 2%, 5% and 10% chicken serum; the low expression of CD14, CD45 and HLA shows that the phenotype is not changed along with the increase of the cell passage number.
The biological characteristics such as cell morphology, proliferation, phenotype and the like are well maintained until the generation P8, which indicates that the chicken serum can be used as an additive for cell culture in vitro and promoting cell growth of cell systems.
And (4) conclusion:
the three groups of test results show that the chicken serum is used as the additive of the cell culture system and is generally suitable for in vitro amplification of primary cells, passage cells and cell lines, and the experimental project provides a new way for in vitro culture of the primary cells, the passage cells and the cell lines.
Claims (2)
1. The culture medium containing chicken serum for in vitro cell culture is characterized by comprising the components of chicken serum and components required by conventional in vitro cell culture.
2. The new application of the chicken serum is characterized in that the chicken serum is used for preparing a culture medium for in vitro cell culture.
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| US4388298A (en) * | 1982-07-14 | 1983-06-14 | The United States Of America As Represented By The Secretary Of Agriculture | Propagation of hemorrhagic enteritis virus in a turkey cell line and vaccine produced |
| JPS6434284A (en) * | 1987-07-29 | 1989-02-03 | Q P Corp | Culture medium for animal cell |
| US20060110824A1 (en) * | 2004-11-24 | 2006-05-25 | Lih-Ren Chen | Method for culturing avian primordial germ cells (PGCs) for a long period and preparing a medium for culturing avian PGCs for a long period |
| US20080124356A1 (en) * | 2003-07-18 | 2008-05-29 | Centre National De La Recherche Scientifique | Keratinocyte Culturing Method and the Use Thereof |
| CN102732476A (en) * | 2012-07-19 | 2012-10-17 | 中国农业大学 | Embryonic stem cell culture medium and application thereof |
| CN104059879A (en) * | 2014-07-04 | 2014-09-24 | 江苏省家禽科学研究所 | Novel method for culturing chicken macrophages HD11 |
| CN105483078A (en) * | 2015-12-24 | 2016-04-13 | 山西农业大学 | Isolation and primary culture methods of chicken small intestinal epithelial cells |
| CN111808801A (en) * | 2020-07-20 | 2020-10-23 | 中国肉类食品综合研究中心 | Method for extracting and culturing pigeon skeletal muscle satellite cells |
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2021
- 2021-12-31 CN CN202111677495.8A patent/CN114292809A/en not_active Withdrawn
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4388298A (en) * | 1982-07-14 | 1983-06-14 | The United States Of America As Represented By The Secretary Of Agriculture | Propagation of hemorrhagic enteritis virus in a turkey cell line and vaccine produced |
| JPS6434284A (en) * | 1987-07-29 | 1989-02-03 | Q P Corp | Culture medium for animal cell |
| US20080124356A1 (en) * | 2003-07-18 | 2008-05-29 | Centre National De La Recherche Scientifique | Keratinocyte Culturing Method and the Use Thereof |
| US20060110824A1 (en) * | 2004-11-24 | 2006-05-25 | Lih-Ren Chen | Method for culturing avian primordial germ cells (PGCs) for a long period and preparing a medium for culturing avian PGCs for a long period |
| CN102732476A (en) * | 2012-07-19 | 2012-10-17 | 中国农业大学 | Embryonic stem cell culture medium and application thereof |
| CN104059879A (en) * | 2014-07-04 | 2014-09-24 | 江苏省家禽科学研究所 | Novel method for culturing chicken macrophages HD11 |
| CN105483078A (en) * | 2015-12-24 | 2016-04-13 | 山西农业大学 | Isolation and primary culture methods of chicken small intestinal epithelial cells |
| CN111808801A (en) * | 2020-07-20 | 2020-10-23 | 中国肉类食品综合研究中心 | Method for extracting and culturing pigeon skeletal muscle satellite cells |
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