CN115044541B - Immortalized pig fat precursor cell line with high differentiation efficiency, and construction method and application thereof - Google Patents
Immortalized pig fat precursor cell line with high differentiation efficiency, and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a high-differentiation-efficiency immortalized pig fat precursor cell line which is preserved in China Center for Type Culture Collection (CCTCC) No. C2022150 in the 5 th month of 2022. Meanwhile, the invention discloses a construction method, a culture and a polyester induction method and application of the cell line. The cell line can be stably passaged and always maintain high differentiation efficiency, and can be used for pig fat precursor cell differentiation research, pig mature fat cell physiological research and cell culture meat production.
Description
Technical Field
The invention relates to the technical field of biology and modern agriculture, in particular to an immortalized pig fat precursor cell line with high differentiation efficiency, and a construction method and application thereof.
Background
Fat is an important flavor substance source of meat, so that the mouthfeel and flavor can be effectively improved by adding a proper amount of fat cells into the cell culture meat. Therefore, the in vitro culture model of the pig fat precursor cells is established, not only can an effective model be provided for researching the growth and development of pig fat tissues, but also can help to research how to improve the production efficiency of pigs, can be used for increasing the flavor of cell culture meat, and has important production and scientific significance.
Currently, most studies and production of porcine fat precursor cells are based on the use of primary cells. However, the number of passages of primary cells is limited, and the primary cells are obtained by continuously slaughtering animals, which is high in cost and inconvenient. There are also reports of establishment of immortalized porcine fat precursor cell lines, but the differentiation efficiency of these cell lines is still rapidly reduced with increasing passage times. At present, no porcine fat precursor cell line capable of reliably maintaining high differentiation efficiency has been established.
Disclosure of Invention
The invention aims to: aiming at the defects and shortcomings of the prior art, the invention provides an immortalized pig fat precursor cell line with high differentiation efficiency, and a construction method and application thereof.
The technical scheme is as follows: the immortalized pig fat precursor cell line with high differentiation efficiency is preserved in China center for type culture collection (CCTCC NO: C2022150) of university of Wuhan in China on 5-month 17 of 2022.
The STR of this cell is characterized by: locus sizes 387A 12F-226+ -1 bp, pigSTR 4C-118+ -1 bp and 122+ -1 bp, pigSTR 5C-262+ -1 bp and 266+ -1 bp, pigSTR 7B-113+ -1 bp, pigSTR 11B-85+ -1 bp, pigSTR 13E-125+ -1 bp and 133+ -1 bp, pigSTR 14A-186+ -1 bp, pigSTR 15A-198+ -1 bp and 202+ -1 bp, pigSTR 17A-155+ -1 bp and 163+ -1 bp, SW 240-93+ -1 bp and 107+ -1 bp, amelogenin-232+ -1 bp and 234+ -1 bp.
The invention relates to a construction method of a high differentiation efficiency immortalized pig fat precursor cell line, which comprises the following steps:
1) Inoculating single cells of the immortalized pig fat precursor cells into a 96-well plate by adopting a limiting dilution method, and culturing until the cells are completely converged in the well;
2) Sequentially culturing cells in each hole from 96-hole plates, 48-hole plates, 24-hole plates and 12-hole plates to 6-hole plates, and carrying out polyester induction differentiation on 1/3 of the cells;
3) Clones with high polyester efficiency were selected, and the remaining 2/3 cells were expanded and stored.
The invention relates to a method for culturing and inducing polyester of immortalized pig fat precursor cell lines with high differentiation efficiency, which comprises the following steps:
1) Culturing in DMEM/F12+10% FBS, and taking the appearance of fibroblast; washing the culture dish with PBS when the cell density approaches 90%, adding pancreatin for digestion for 3min, terminating pancreatin reaction with DMEM/F12+10% FBS, and passaging 30% cells to a new culture dish;
2) After the cells reached full confluence, a large number of lipid droplets were observed by culturing for 3d using induction medium and maintaining the medium for 4 d.
The formula of the induction culture medium is as follows: DMEM/F12, 10% FBS,1ug/mL Insulin,5nM T3, 100uM IBMX,2ug/mL Dexamethasone,125uM Indomethacin,1uM Rosiglitazone,33uM Biotin, 17uM Pantothenic Acid,1ug/uL transferin;
the formula of the maintenance culture medium is as follows: DMEM/F12, 10% FBS,1ug/mL instrument, 5nM T3,1uM Rosiglitazone.
The method for culturing and inducing polyester of immortalized pig fat precursor cell line with high differentiation efficiency comprises the following steps:
culturing immortalized pig fat precursor cells;
obtaining monoclonal cells by a limiting dilution method;
inducing differentiation of the obtained monoclonal cells;
screening out the monoclonal cell line with highest polyester differentiation efficiency;
the monoclonal with highest polyester differentiation efficiency is Pig#4. And preserving the same.
And the serial passage and differentiation performance of the obtained monoclonal cell line are identified.
The invention relates to application of immortalized pig fat precursor cell line with high differentiation efficiency in food development.
The invention relates to application of immortalized pig fat precursor cell line with high differentiation efficiency in virus culture or detection.
The invention relates to application of immortalized pig fat precursor cell line with high differentiation efficiency in vaccine development and production.
The invention relates to application of immortalized pig fat precursor cell line with high differentiation efficiency in drug screening.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the invention constructs the pig fat precursor cell line which can be stably passed for more than 35 generations and always keeps high differentiation efficiency for the first time through a great amount of creative labor of the inventor: pig#4. By optimizing, a culture solution formula capable of exerting the differentiation capacity of the Pic #4 polyester to the maximum is obtained.
The immortalized Pig fat precursor cell line Pic#4 with high differentiation efficiency can be stably passaged, so that the use of primary Pig fat cells is reduced, and the production and experiment cost is reduced; pig#4 can always maintain extremely high differentiation capacity, can be used for researching a polyester mechanism for differentiating fat precursor cells, and can also be used for producing cell culture meat.
The immortalized pig fat precursor cell line with high differentiation efficiency has been passaged for more than 35 generations so far; the use of 9 factor induction medium can allow more than 90% of its cells to become mature adipocytes with large numbers of lipid droplets. The cell line can be used for pig fat precursor cell differentiation research, pig mature fat cell physiological research and cell culture meat production.
Drawings
FIG. 1 is a morphology of 107 monoclonal porcine fat precursor cells;
FIG. 2 is a graph of 86 strain of monoclonal oil red O staining;
FIG. 3 is a graph of OD values of monoclonal oil red staining;
FIG. 4 is a graph of the growth of Pic #4 at different generations;
FIG. 5 is a graph showing the effect of changing insulin concentration in induction medium on differentiation; FIG. A is a cell culture diagram; panel B is a graph of concentration versus differentiation effect;
FIG. 6 is a graph showing the effect of changing IBMX concentration of induction medium on differentiation; FIG. A is a cell culture diagram; panel B is a graph of concentration versus differentiation effect;
FIG. 7 is a graph showing the effect of varying dexamethasone concentration in the induction culture medium on differentiation; FIG. A is a cell culture diagram; panel B is a graph of concentration versus differentiation effect;
FIG. 8 is a graph showing the effect of changing indomethacin concentration of induction medium on differentiation; FIG. A is a cell culture diagram; panel B is a graph of concentration versus differentiation effect;
FIG. 9 is a graph of polyester differentiation of different algebraic Pic #4 cells; FIG. A is a cell culture diagram; FIG. B is a differentiation chart;
FIG. 10 is a graph of FIG. 1 showing the results of PIg#4STR locus detection;
FIG. 11 is a graph of FIG. 2 showing the results of PIg#4STR locus detection;
FIG. 12 is a graph of FIG. 3 showing the results of PIg#4STR locus detection;
FIG. 13 is a graph of the results of PIg#4STR locus detection of FIG. 4;
FIG. 14 is a graph of the results of PIg#4STR locus detection FIG. 5;
FIG. 15 is a graph of the results of PIg#4STR locus detection of FIG. 6;
FIG. 16 is a graph of the results of PIg#4STR locus detection FIG. 7;
FIG. 17 is a graph of FIG. 8 showing the results of PIg#4STR locus detection;
FIG. 18 is a graph of FIG. 9 showing the results of PIg#4STR locus detection;
FIG. 19 is a graph of the results of PIg#4STR locus detection 10;
FIG. 20 is a graph of the results of PIg#4STR locus detection FIG. 11.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and the specific embodiments.
Immortalized porcine fat precursor cells employed in the present invention were established by the inventors themselves.
Example 1: the monoclonal cell line was obtained by limiting dilution.
1. Immortalized porcine fat precursor cells were seeded in 96-well plates at a density of 0.5 cells per well;
2. cell status was observed daily and when cells reached 90% confluence they were passaged to 48 well plates;
3. observing the cells in the step 2, and passaging the cells to a 24-well plate when the cells reach 90% confluence;
4. observing the cells in the step 3, and passaging the cells to a 12-well plate when the cells reach 90% confluence;
5. observing the cells in the step 4, and passaging the cells to a 6-well plate when the cells reach 90% confluence;
results: a total of 107 single clones were obtained, see FIG. 1.
Example 2: screening monoclonal with highest differentiation induction efficiency.
1. Cells in the 6-well plate of example 1, step 5, were digested with pancreatin, 2/3 of the cells were frozen in liquid nitrogen, and 1/3 of the cells were passaged into one well of a 12-well plate;
2. after the monoclonal cells in the 12-well plate grow to be completely converged;
2. changing the culture solution in the step 2 into an induction culture solution, and placing the culture solution in a cell culture box for culturing for 3 days;
3. the culture solution in the step 2 is replaced by a maintenance culture solution, and the culture solution is replaced every 2 days for 2 times.
4. The cells in step 3 were fixed with 4% paraformaldehyde, photographed with an oil red O stain, and absorbance at 500nm was measured using a spectrophotometer.
The induction culture solution comprises the following components: 1. Mu.g/mL of instrument, 5nM T3, 100. Mu.M IBMX, 2. Mu.g/mL Dexamethasone, 125. Mu.M Indomethacin, 1. Mu.M Rosilitazone, 33. Mu.M Biotin, 17. Mu. M Pantothenic Acid, 1. Mu.g/uL of Transferrin were added to DMEM/F12 complete medium containing 10% FBS.
The maintenance culture solution comprises the following components: 1. Mu.g/mL of instrument, 5nM T3, 1. Mu.M Roskilite was added to DMEM/F12 complete medium containing 10% FBS.
Oil red O staining identification: after the differentiation of the fat precursor cells is completed, the maintenance cell culture solution in each hole is discarded, 4% paraformaldehyde is added, and the cells are fixed for 15min; washing with 60% isopropanol once; drying at room temperature; adding 1mL of oil red O working solution into each hole, and standing at room temperature for 20min; rapidly discarding the oil red O working solution in each hole, and cleaning for 4 times by deionized water; standing and drying at room temperature, and then taking an oil red O dyeing photo; 1.5mL of isopropanol is added into each hole, and the mixture is incubated for 20min at room temperature; sucking the supernatant; the absorbance at 500nm was measured using a spectrophotometer.
Results: the cells were partially removed and died during differentiation, and a total of 85 single clones were induced to differentiate, of which 60 single clones had differentiation ability, see FIG. 2.
The OD500 value of the single clone with obvious oil red staining is measured, and the single clone is ranked according to the differentiation capacity, and the single clone is shown in figure 3. Clone4 was found to have the highest differentiation efficiency and was designated Pic #4.
Example 3: proliferation activity assay for Pic #4.
1. Pig #4 generation 10 (P10), generation 20 (P20), generation 30 (P30)) Immortalized porcine fat precursor cells generation 10 (PSPA P10) at 2.5 x10 4 The individual/well was seeded in 24-well plates.
2. Placing at 37deg.C, 5% CO 2 Culturing in a cell culture incubator.
3. Cells were digested with pancreatin every 24 hours and counted using a hemocytometer.
4. The inoculation time was taken as the first day (1 d), the incubation time (d) was taken as the abscissa, the number of cells (x 10) 4 ) The ordinate is plotted.
Results: the 10 th, 20 th, 30 th generation of Pig #4 and 10 th generation of immortalized Pig fat precursor cells had the same growth trend, see fig. 6. The cells grow slowly at about 48 hours before cell inoculation, the cell number starts to multiply at day 3, the cell increases in speed to start to slow down at day 6, and the cells are inhibited in contact at day 9, and the cells almost stop growing. Pig #4 was shown to have proliferative capacity consistent with immortalized porcine fat precursor cells and Pig #4 remained proliferative when passaged over 30 passages.
Example 4: the optimum medium formulation for the differentiation of Pic #4 polyester was determined.
1. On the basis of the induction medium formulation of example 2, step 4, the concentration of each component was varied one by one as follows:
Insulin:0.35ug/mL,0.7ug/mL,1ug/mL,3.5ug/mL；
IBMX:0.05mM，0.1mM，0.25mM，0.5mM；
Dexamethasone:0.5ug/mL，0.66ug/mL，1ug/mL，2ug/mL；
Indomethacin:125uM，62.5uM，0uM；
preparing an induction culture medium;
2. pic #4 was passaged to 12-well plates and after cell growth confluency, the cells were cultured for 3 days using the induction medium described above.
3. Changing the induction culture medium in the step 2 into a maintenance culture solution for culturing for 4 days, changing the solution every 48 hours, wherein the maintenance culture solution comprises the following components: 1. Mu.g/mL of instrument, 5nM T3, 1. Mu.M Roskilite was added to DMEM/F12 complete medium containing 10% FBS.
4. The cells in step 3 were fixed with 4% paraformaldehyde, photographed with an oil red O stain, and absorbance at 500nm was measured using a spectrophotometer.
Results: the optimal concentration of insulin is 1ug/mL, shown in figure 5, the optimal concentration of IBMX is 100uM, shown in figure 6, the optimal concentration of dexamethasone is 2ug/mL, shown in figure 7, and the optimal concentration of indomethacin is 125uM, shown in figure 8.
Example 5: different algebraic Pic #4 polyesters were tested for their ability to differentiate.
1. Pig #4 generation 10 (P10), generation 20 (P20), generation 30 (P30) was added at 2.5 x10 4 The cells were seeded in 24-well plates and placed at 37℃in 5% CO 2 Culturing in a cell culture incubator.
2. After the cells are converged, the culture solution in the step 1 is replaced by an induction culture solution, and the induction culture solution is placed in a cell culture box for 3 days;
3. the culture solution in the step 2 is replaced by a maintenance culture solution, and the culture solution is replaced every 2 days for 2 times.
The induction medium and maintenance medium formulations were the same as in example 2.
4. The cells in step 3 were fixed with 4% paraformaldehyde, photographed with an oil red O stain, and absorbance at 500nm was measured using a spectrophotometer.
Results: pic #4 at generation 10, 20 and 30 has similar polyester differentiation ability, as shown in FIG. 9.
Example 6: STR characterization of pin #4.
STR (short repeat sequence short tandem repeats) is a special sequence that is widely found in the genome and is characterized by the presence of multiple base consecutive repeats. The number of such repeats is highly genetic diverse and therefore the STR combinations are not identical for each individual. STR detection has been widely used for paternity testing and forensic personal gene fingerprinting.
The pig STR detection method used in this example is disclosed in the "construction of a multiplex amplification system for 12 STR loci in pigs" text (DOI: 10.16467/j.1008-3650.2019.04.006).
1. The cells were cultured until confluent and genomic DNA was extracted using the Ezup column animal tissue genomic DNA extraction kit.
2. The PCR system was configured as follows:
the primer sequences used and modifications thereof are as follows:
3. PCR amplification was performed as follows:
4. STR detection
4.1 a 96 well reaction plate was used and the plate name and date of the experiment were marked with a marker.
And 4.2, manufacturing an electronic STR detection table, and automatically generating an on-machine table.
4.3 Using a continuous applicator, 990. Mu.l of a mixture of HIDI and 10ul of LIZ500 was pipetted into a 96 well reaction plate with 10. Mu.l per well.
4.4 96-well plates were placed in a plate centrifuge and centrifuged at 1200rmp for 15s.
4.5 Using a 12-row 10. Mu.L gun, 1. Mu.L of the PCR product was added to the wells corresponding to the 96-well plates against the STR assay table.
4.6 the 96-well plates were placed in a plate centrifuge and centrifuged at 1200rmp for 15s.
4.7 sealing the 96-well plate with a sealing plate membrane, shaking, and placing the 96-well plate in a flat plate centrifuge for 30s by centrifugation at 1200 rmp. Placed in a PCR instrument.
4.8 denaturation procedure at 98℃for 5min without heating the hot lid, immediately after the procedure was completed the 96-well plate was placed on an ice-water mix and rapidly cooled.
4.9 the 96-well plates were placed in a plate centrifuge and centrifuged at 1200rmp for 15s.
4.10 STR samples were tested using the ABI 3730xl apparatus.
4.11 the number of bases of the peak was read based on the off-chip data.
Through detection, the immortalized Pig fat precursor cell line Pic#4 with high differentiation efficiency can be stably passaged, so that the use of primary Pig fat cells is reduced, and the production and experiment cost is reduced; pig#4 can always maintain extremely high differentiation capacity, can be used for researching a polyester mechanism of fat precursor cell differentiation, and can also be used for producing cell culture meat.
Claims (2)
1. An immortalized pig fat precursor cell line with high differentiation efficiency is preserved in China Center for Type Culture Collection (CCTCC) No. C2022150 at 5-17 of 2022.
2. The high differentiation efficiency immortalized porcine fat precursor cell line according to claim 1, wherein: the STR of this cell is characterized by: locus sizes 387A 12F-226+ -1 bp, pigSTR 4C-118+ -1 bp and 122+ -1 bp, pigSTR 5C-262+ -1 bp and 266+ -1 bp, pigSTR 7B-113+ -1 bp, pigSTR 11B-85+ -1 bp, pigSTR 13E-125+ -1 bp and 133+ -1 bp, pigSTR 14A-186+ -1 bp, pigSTR 15A-198+ -1 bp and 202+ -1 bp, pigSTR 17A-155+ -1 bp and 163+ -1 bp, SW 240-93+ -1 bp and 107+ -1 bp, amelogenin-232+ -1 bp and 234+ -1 bp.
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