CN117487745B - Pigeon fat precursor cell separation and in-vitro culture method, culture and application - Google Patents
Pigeon fat precursor cell separation and in-vitro culture method, culture and application Download PDFInfo
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Abstract
The invention discloses a method for separating and culturing pigeon fat precursor cells in vitro and a culture and application thereof, which relate to the technical field of animal cell extraction, and the method for separating pigeon fat precursor cells is a combination of a mechanical method and a collagenase digestion method, and digestion is stopped by a basic culture medium DMEM containing 20% fetal bovine serum so as to control digestion time, so that pigeon subcutaneous fat meat emulsion can be digested and extracted within a target range; the separation and in-vitro culture of the pigeon fat precursor cells for cell culture can be realized under simpler and more efficient conditions, centrifugation is not needed, erythrocyte lysate is not needed, the steps are simple and easy to operate, and the cost is low; the identification by a cell immunofluorescence method proves that the pigeon fat precursor cells are indeed obtained; the differentiation efficiency of the obtained fat precursor cells is high, the differentiation is uniform and the stability is good through cell differentiation identification.
Description
Technical Field
The invention relates to the technical field of animal cell extraction, in particular to a pigeon fat precursor cell separation and in-vitro culture method, a culture and application.
Background
With the increasing interest in sustainable foods and animal welfare, biological breeding meats are receiving increasing attention as a potential alternative. The biological cultured meat is a novel meat food which is prepared by controlling the rapid proliferation, directional differentiation and collecting and processing of animal cells in an in vitro culture mode.
The importance of seed cell extraction is that it is the basis for meat cultivation. By extracting seed cells, a small fraction of the cell sample can be obtained and by culturing and expanding these cells, a large scale meat production is achieved. This approach has many advantages over traditional farming methods, including reduced reliance on animals, improved food safety, reduced environmental burden, etc. Seed cell extraction can also help solve some of the problems faced by traditional farming, such as resource consumption, animal welfare, and climate change challenges.
The key to seed cell extraction is to select cells with high differentiation potential and ensure that they retain their multipotency and proliferative capacity during culture. For example, skeletal muscle satellite cells (muscle seed cells) may differentiate into muscle cells, fat precursor cells (fat seed cells) into fat cells, etc., thereby achieving the cultivation of tissues and mouthfeel similar to conventional meats.
Although seed cell extraction is important in the process of cell culturing meat, further research and technological development are needed to improve the efficiency and cost effectiveness of meat culture. The method comprises the steps of improving the extraction and culture method of seed cells, optimizing the formula and environmental conditions of a culture medium, solving the challenges of large-scale production and market popularization of cultured meat and the like, and aiming at the diversification of human edible animals, different treatment modes for extracting seed cells of cultured meat of different animals are provided, including seed cell extraction positions, extraction ages, treatment means and the like, so that the extraction of seed cells still has a larger research space.
Along with the progress of scientific technology, the contribution of cell culture meat to sustainable food production and human health in the future can be expected, pigeon meat is popular meat, but in view of the breeding difficulty of pigeons, the eating surface of pigeons is far less than that of poultry such as chickens, ducks and geese, and the appearance of biological culture meat is expected to solve the problem, although some methods are used for extracting fat precursor cells of pigeons at present, centrifuge equipment is needed in the extraction process, cells are damaged in the centrifugation process, and cell activity is reduced and dead cells are increased. Therefore, there is a need for a more efficient and viable method to overcome these technical problems, thereby extracting high quality pigeon fat precursor cells as seed cells for biological breeding of meat.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for separating and in-vitro culturing pigeon fat precursor cells, a culture and application thereof, which are used for solving the technical problem that the cells are damaged in the centrifugation process in the traditional pigeon fat precursor cell extraction method, and the extracted cells have better differentiation potential and can meet the differentiation requirement of biological cultured meat seed cells.
The technical scheme adopted by the invention is as follows:
the method for separating pigeon fat precursor cells comprises the following steps:
(1) Cutting subcutaneous fat of a pigeon which has just been subjected to shell cutting into minced meat, adding collagenase digestion liquid with the volume of 3-5 times that of the minced meat, placing the minced meat in a 37 ℃ incubator, digesting for 30-60 min, and uniformly mixing the minced meat upside down every 10-20 min; adding a basic culture medium DMEM containing 10-20% fetal bovine serum, which is 8-10 times of the collagenase digestive juice, stopping digestion, and filtering by using a cell sieve with 70-150 mu m;
(2) Directly paving the filtrate obtained by filtration to a cell culture dish, and placing the cell culture dish in an incubator for culture at the culture temperature of 37-41 ℃;
(3) Culturing for 2-3 days, discarding the old culture medium, washing gently with PBS, and then adding a basic culture medium DMEM containing 10-20% fetal calf serum to obtain the culture.
Further, the collagenase in the step (1) is type I collagenase, the concentration of the collagenase digestion liquid is 1-2 mg/mL, and the collagenase digestion liquid is prepared by adopting Hanks liquid containing calcium and magnesium as a solvent.
Further, the cell screen of step (1) is a 100 μm disposable cell filter screen.
Further, the fetal calf serum in the step (1) and the step (3) is Australian fetal calf serum, and the fetal calf serum accounts for 20% of the volume of the basic DMEM culture medium.
Further, the culture temperature in the step (2) was 41 ℃.
A culture containing fat precursor cells obtained by the method of isolation and in vitro culture of pigeon fat precursor cells according to any one of the above.
More preferably, the morphology and identity identification method of pigeon fat precursor cells in the culture are respectively a phalloidin staining method and a cellular immunofluorescence staining method.
More preferably, the identification method of the differentiation potential of pigeon fat precursor cells in the culture specifically comprises the following steps:
(1) Inoculating the culture into a cell culture plate for two-dimensional culture;
(2) When the cell confluence is 90% -100%, the complete culture medium is changed into a differentiation culture medium, and the differentiation culture is induced to be 2% -6 d; the components in the differentiation culture medium comprise a basal culture medium DMEM, a adipogenic differentiation induction factor and fetal bovine serum;
(3) Changing differentiation medium every 2 d;
(4) Discarding the differentiation medium, slowly adding PBS along the cell culture plate wall for soft rinsing;
(5) Adding a cell fixing solution, fixing for 15-30 min, and rinsing with PBS;
(6) Adding 5-10 mu mol/L BODIPY working solution and 1-2 mu g/mL DAPI solution, and dyeing for 10-15 min in a dark place;
(7) Discarding the dyeing liquid, rinsing with PBS, and directly soaking for 5 min without shaking;
(8) Observed using a fluorescence microscope and photographed.
The culture is used as seed cells in the process of preparing pigeon meat.
The culture is applied to in vitro research materials for researching the fat development mechanism of pigeons.
In summary, compared with the prior art, the invention has the following advantages and beneficial effects:
1. the separation method of pigeon fat precursor cells is a combination of a mechanical method and a collagenase digestion method, and digestion is stopped by a basic culture medium DMEM containing 20% fetal calf serum so as to control digestion time, so that pigeon subcutaneous fat meat emulsion can be digested and extracted within a target range; the separation and in-vitro culture of the pigeon fat precursor cells for cell culture can be realized under simpler and more efficient conditions, centrifugation is not needed, erythrocyte lysate is not needed, the steps are simple and easy to operate, and the cost is low; the identification by a cell immunofluorescence method proves that the pigeon fat precursor cells are indeed obtained; the differentiation efficiency of the obtained fat precursor cells is high, the differentiation is uniform and the stability is good through cell differentiation identification;
2. the invention can provide better tools and methods for livestock/poultry industry, biomedical research and biological cultivation meat research, further understand the development process of pigeon fat cells, and can be used for regulating and controlling fat development and metabolism and physiological and pathological processes related to the fat development and metabolism.
Drawings
FIG. 1 is a graph showing the comparison of the numbers of pigeon fat precursor cells attached by centrifugation and without centrifugation;
FIG. 2 is a graph showing the results of staining the cytoskeleton and nuclei of pigeon fat precursor cells;
FIG. 3 shows the immunofluorescence staining identification result of pigeon fat precursor cells;
FIG. 4 is a graph showing the effect of different temperatures on pigeon fat precursor cell growth;
FIG. 5 shows the results of in vitro differential staining of pigeon fat precursor cells.
Detailed Description
The advantages and various effects of the present invention will be more clearly apparent from the following detailed description and examples. It will be understood by those skilled in the art that these specific embodiments and examples are intended to illustrate the invention, not to limit the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Unless otherwise specifically indicated, the various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or may be prepared by existing methods.
The present application will be described in detail with reference to examples and experimental data.
Example 1 this example uses ophthalmic scissors to cut the subcutaneous fat of a pigeon that has just emerged into 1 mm 3 Then adding a type I collagenase solution for digestion, then screening cells, directly paving the obtained mixed solution on a cell culture dish, and attaching to the wall to obtain pigeon fat precursor cells. It was found by microscopic observation that fat precursor cells of pigeons were irregularly shaped, polygonal, short spindle-shaped, etc., and that the number of obtained fat precursor cells was significantly increased compared with the centrifugation (fig. 1).
The specific separation steps of the pigeon fat precursor cells are as follows:
(1) Killing pigeons which just come out of the shells;
(2) Transferring pigeons into a biosafety cabinet subjected to ultraviolet sterilization treatment, and wiping the whole body with 75% alcohol for sterilization;
(3) Separating abdominal subcutaneous fat using a sterile scalpel, an ophthalmic scissors and an ophthalmic forceps;
(4) The subcutaneous fat is firstly rinsed in 75% alcohol for 1 time and then transferred into PBS solution;
(5) Removing macroscopic blood vessels, muscle and other impurities to obtain subcutaneous fat of pigeon, and cutting the subcutaneous fat into pieces of about 1 mm 3 Ground meat emulsion of size;
(6) Adding 4 times volume of 2 mg/mL type I collagenase solution into a centrifuge tube containing minced meat; transferring to a centrifuge tube;
(7) Placing the centrifuge tube in a 37 ℃ incubator, digesting for 30-60 min, and manually reversing every 10 min;
(8) Adding 8-10 equal volumes of complete culture medium (DMEM containing 20% fetal bovine serum) to stop digestion, and filtering with a 100 μm cell sieve;
(9) Directly spreading the digestive juice to a cell culture dish of 10 cm, and placing the cell culture dish in an incubator for culturing at 41 ℃;
(10) Culturing for 2-3 days, discarding the old culture medium, washing gently with PBS, and then adding a basic DMEM culture medium containing 10-20% fetal calf serum to obtain the culture.
In the embodiment, two methods, namely a phalloidin staining method and a cellular immunofluorescence method, are adopted to carry out cell morphology characterization and cell identity characterization on cells in the respective cultures. The phalloidin is a cytoskeletal staining device, can clearly show the morphology and distribution of intracellular microfilaments, and can assist in observing the cell morphology.
The cells in the culture were morphological characterized by the phalloidin staining technique as follows:
(1) The first day, cells were seeded into 24-well cell culture plates;
(2) The next day, the medium was discarded, washed 3 times with PBS, and 500. Mu.L of cell fixative was added to the wells and fixed for 15-30 min. Discarding the fixing solution, washing with PBS for 3 times, each time for 5 min;
(3) Adding 0.1-0.5% of triton X-100 to perform cell permeation for 5-10 min, discarding permeation liquid, and washing with PBS for 1-2 times, each time for 5 min;
(4) Adding 200-300 mu L of FITC-labeled phalloidin working solution, diluting the phalloidin by 1% BSA (200:1), and dyeing for 30-40 min at room temperature;
(5) Adding 200-300 mu L of 2 mu g/ml DAPI staining solution into the hole to stain the cell nucleus;
(6) PBS is washed for 2-3 times, each time for 5 min, and then a fluorescence microscope is used for observation and photographing.
As a result, as shown in FIG. 2, the obtained pigeon fat precursor cells were stained with phalloidin and DAPI to more clearly characterize the cell morphology, which indicates that the present example successfully isolated pigeon fat precursor cells.
The steps of using cellular immunofluorescence techniques to characterize the identity of cells in culture are as follows:
(1) The first day, cells were seeded into 24-well cell culture plates;
(2) The next day, the culture medium is discarded, the culture medium is washed for 3 times by PBS, 300-500 mu L of cell fixing solution is added into the holes, and the cells are fixed for 15-30 min. Discarding the fixing solution, washing with PBS for 3 times, each time for 5 min;
(3) Adding 0.1% -0.5% triton X-100 for cell permeation for 15-20 min, discarding permeation liquid, and washing with PBS for 3 times, each time for 5 min;
(4) Adding 300-500 mu L of 0.2% BSA, and sealing for 45-60 min at room temperature;
(5) The blocking solution is discarded, 200-300 mu L of rabbit-sourced DLK1 primary antibody (dilution ratio 1:50-1:100) is added, and the mixture is left at 4 ℃ overnight. Washing with PBS for 3 times, each time for 5 min;
(6) 200-300 mu L of goat anti-rabbit secondary antibody (dilution ratio is 1:100-1:200) is added, and the temperature is 4 ℃ overnight;
(7) Washing with PBS for 3 times, each time for 5 min, and adding 200-300 mu L of DAPI (1-2 mu g/mL) staining solution to stain cell nuclei;
(8) PBS was washed 3 times for 5 min each, after which it was observed with a fluorescence microscope and photographed.
As shown in FIG. 3, the obtained fat precursor cells were identified by immunofluorescence technique, and it was found that almost all cells expressed DLK1 protein, which indicates that the present example successfully isolated pigeon fat precursor cells.
Example 2 this example is a study of the effect of in vitro culture temperature on the number of pigeon fat precursor cells.
The culture temperature in step (9) of the specific isolation procedure of pigeon fat precursor cells of example 1 was replaced with 37℃and compared with the number of fat precursor cells obtained in example 1 by observation under a microscope, and the results of the study are shown in FIG. 4, it is clear that 41℃in vitro culture can give a greater number of pigeon fat precursor cells, indicating that 41℃is advantageous for proliferation of fat precursor cells.
Example 3 this example provides the identification of the cell differentiation properties of the resulting pigeon fat precursor cells:
1. cell differentiation culture of fat precursor cells
(1) Inoculating fat precursor cells into a cell culture plate or a culture dish for two-dimensional culture;
(2) When the cell confluency is 90% -100%, the complete culture medium is changed into a lipid-forming induced differentiation culture medium, and induced differentiation culture is 6 d, wherein the differentiation culture medium comprises a basic culture medium DMEM, lipid-forming differentiation induction factors (5 ug/ml insulin, 150 mu mol/L oleic acid) and 10% fetal bovine serum;
(3) The adipogenic differentiation medium is changed every 2 d;
2. differentiation identification of pigeon adipocytes
Generally, the method comprises an oil red O staining method, a BODIPY staining method and the like, and the embodiment provides the BODIPY staining method, wherein BODIPY is a near infrared short-wavelength fluorescent dye which can specifically act on oil drops composed of neutral lipids, and the specific operation is as follows:
(1) The culture medium is discarded, the cells after differentiation are easily detached from the wall by external impact force after being gently rinsed 1 time by PBS, and the cells are preferably slowly added along the wall of a cell culture dish or a culture plate;
(2) Adding 300-500 mu L of cell fixing solution, fixing for 15-30 min, and rinsing 3 times with PBS;
(3) Adding 300-500 mu L of 5-10 mu mol/L BODIPY working solution and 1-2 mu g/mL DAPI solution, and dyeing for 10-15 min in a dark place;
(4) Discarding the dyeing liquid, rinsing with PBS for 3 times, each time for 5 min, and directly soaking for 5 min during washing without shaking table or manual shaking;
(5) Observing and photographing under a fluorescence microscope;
as shown in FIG. 5, it was observed that the cells differentiated by 6 d had vacuolated lipid droplets, and after staining, these lipid droplets were stained green with BODIPY, indicating that our isolated pigeon fat precursor cells could differentiate into mature adipocytes, and that the isolated pigeon fat precursor cells had high differentiation potential and could be used as seed cells for biological growth of meat.
The foregoing examples merely represent specific embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the present application. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present application, which fall within the protection scope of the present application.
Claims (5)
1. The method for separating and culturing pigeon fat precursor cells in vitro is characterized by comprising the following steps:
(1) Cutting subcutaneous fat of a pigeon which has just been subjected to shell cutting into minced meat, adding collagenase digestion liquid with the volume of 3-5 times that of the minced meat, wherein the collagenase is type I collagenase, placing the minced meat in a 37 ℃ incubator, digesting for 30-60 min, and uniformly mixing the minced meat upside down every 10-20 min; adding a basic culture medium DMEM containing 10-20% fetal bovine serum, which is 8-10 times of the collagenase digestive juice, stopping digestion, and filtering by using a cell sieve with 70-150 mu m;
(2) Directly paving the filtrate obtained by filtration to a cell culture dish, and placing the cell culture dish in an incubator for culture at the culture temperature of 37-41 ℃;
(3) Culturing for 2-3 days, discarding the old culture medium, washing gently with PBS, and then adding a basic culture medium DMEM containing 10-20% fetal calf serum to obtain a culture containing fat precursor cells.
2. The method for separating and culturing pigeon fat precursor cells in vitro according to claim 1, wherein the concentration of the collagenase digestion solution in the step (1) is 1-2 mg/mL, and the collagenase digestion solution is prepared by using Hanks solution containing calcium and magnesium as a solvent.
3. The method for isolating and culturing pigeon fat precursor cells in vitro according to claim 1, wherein the cell sieve in step (1) is a disposable cell filter sieve of 100 μm.
4. The method for isolating and culturing pigeon fat precursor cells in vitro according to claim 1, wherein the fetal bovine serum in step (1) and step (3) is australian fetal bovine serum, and the fetal bovine serum is 20% by volume of DMEM basal medium.
5. The method for isolating and culturing pigeon fat precursor cells in vitro according to claim 1, wherein the culturing temperature in step (2) is 41 ℃.
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