CN113174367A - Separation method of preadipocytes - Google Patents

Separation method of preadipocytes Download PDF

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CN113174367A
CN113174367A CN202110381028.4A CN202110381028A CN113174367A CN 113174367 A CN113174367 A CN 113174367A CN 202110381028 A CN202110381028 A CN 202110381028A CN 113174367 A CN113174367 A CN 113174367A
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preadipocytes
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fat
collagenase
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万荣
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Baise University
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
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Abstract

The invention relates to a method for separating preadipocytes, in particular to a method for preparing preadipocytes, which comprises the following steps: (1) mixing fat and a collagenase I solution, and then carrying out incubation digestion to obtain a digestive juice; (2) neutralizing the digestive juice with a neutralizing solution, and filtering to obtain a filtrate; (3) and centrifuging the filtrate, and collecting the precipitate to obtain the preadipocytes. The preadipocytes prepared by the method have strong activity, the growth curve of in vitro culture is similar to S type, the preadipocytes conform to the normal growth and proliferation rule of in vitro culture cells, the ploidy of chromosomes is normal after continuous passage, the basic genetic characteristics of normal cells are kept, and the preadipocytes have excellent in vitro adipogenic capacity after induced differentiation.

Description

Separation method of preadipocytes
Technical Field
The invention relates to the technical field of biology, in particular to a method for separating preadipocytes.
Background
Adipocytes are derived from the stepwise differentiation and development of mesoderm-derived multipotent Mesenchymal Stem Cells (MSCs), and the differentiation and development process is roughly as follows: MSC directionally differentiating to fat cell to form adipocyte (adipoblast); ② the adipocyte is formed into preadipocyte (preadipocyte) through growth inhibition and cell proliferation; ③ forming a multi-chamber fat cell (multi-chamber cell) by the preadipocyte through the growth inhibition, the cell proliferation and the change of a series of gene expression, wherein the multi-chamber fat cell contains a large number of small fat droplets; the multi-chambered fat cell gradually collects into a large fat drop along with the deposition of lipid in the cell, fills most of the fat cell, forms a single-chambered fat cell (unilocular cell), and is a mature fat cell.
Preadipocytes (Preadipocytes) are induced to accumulate lipid droplets within the cytoplasm and differentiate into mature adipocytes. Preadipocytes can be used for studying the mechanisms of fat development and fat deposition, and can also be applied to the technical development of plastic departments in the field of human medicine, for example, preadipocytes are used for beauty after being used for obtaining and purifying fat tissues.
In the prior art, preadipocytes are often extracted from adipose tissues, however, the existing preadipocyte extraction method has a plurality of defects, such as poor activity of the preadipocytes obtained by extraction, slow in-vitro growth, non-conformity with the normal growth and proliferation rule of in-vitro cultured cells, abnormal ploidy of chromosomes after continuous passage, difficulty in retaining the basic genetic characteristics of normal cells, poor adipogenic capability after induced differentiation and the like. Therefore, how to develop a preadipocyte preparation method which has strong activity, retains the basic genetic characteristics of normal cells after subculture and has the capability of inducing adipogenesis becomes a hotspot of current research.
Therefore, there is a need in the art to develop a method for preparing preadipocytes that have high activity, retain the basic genetic characteristics of normal cells after subculture, and have the ability to induce adipogenesis.
Disclosure of Invention
The invention aims to provide a preadipocyte preparation method which has strong activity, retains the basic genetic characteristics and the adipogenic induction capability of normal cells after subculture.
In a first aspect, the present invention provides a method for preparing preadipocytes, the method comprising the steps of:
(1) mixing fat and a collagenase I solution, and then carrying out incubation digestion to obtain a digestive juice;
(2) neutralizing the digestive juice with a neutralizing solution, and filtering to obtain a filtrate;
(3) and centrifuging the filtrate, and collecting the precipitate to obtain the preadipocytes.
Preferably, the preadipocytes are selected from the group consisting of: intramuscular preadipocytes, subcutaneous preadipocytes, visceral preadipocytes, or a combination thereof.
Preferably, in step (1), the fat is derived from a human or non-human mammal.
Preferably, the non-human mammal is a cow, sheep, dog, pig, horse, rabbit or cat.
Preferably, in step (1), the fat is selected from the group consisting of: intramuscular fat, subcutaneous fat, visceral fat, or a combination thereof.
Preferably, in the step (1), the fat comprises a crushed (or cut) fat.
In another preferred embodiment, the comminuted fat comprises fat pieces.
Preferably, the fat mass is 0.2-2mm in size3Preferably 0.5-1.5mm3Preferably 0.8-1.2mm3Preferably 1mm3。
Preferably, in step (1), the weight ratio of said fat to said collagenase type I solution is 0.5-1.5:0.5-1.5, preferably 0.8-1.2:0.8-1.2, more preferably 1: 1.
Preferably, in step (1), collagenase type I is contained in the collagenase type I solution in an amount of 0.5-1mg/mL, preferably 0.8mg/mL, more preferably 1 mg/mL.
Preferably, in the step (1), the collagenase type i solution is prepared by the following method:
weighing type I collagenase, dissolving the type I collagenase in DPBS, and adjusting the pH value to 7.2-7.4 to obtain a type I collagenase solution.
Preferably, in the step (1), the collagenase type i solution is prepared by the following method:
weighing collagenase type I, dissolving the collagenase type I in DPBS, adjusting the pH value to 7.2-7.4, and filtering to obtain collagenase type I solution.
Preferably, the filtration is a 0.22 μm membrane filtration.
Preferably, in the step (1), the fat comprises washed fat.
Preferably, the washed fat comprises buffer-washed fat.
Preferably, the buffer comprises a DPBS buffer solution.
Preferably, in the step (1), the temperature of the incubation digestion is 20-40 ℃, preferably 30-37 ℃, and more preferably 37 ℃.
Preferably, in the step (1), the incubation digestion time is 0.5-1.5h, preferably 0.8-1.2h, and more preferably 1 h.
Preferably, in the step (1), the mixture is shaken every 4-6min for 1 time during the incubation digestion.
Preferably, in the step (2), the neutralization solution comprises a basic culture medium.
Preferably, in the step (2), the neutralizing solution comprises a basic medium added with FBS.
Preferably, the basal medium comprises DMEM basal medium.
Preferably, in the basic medium added with FBS, the content of the FBS is 5-15 v/v%, preferably 8-12 v/v%, and more preferably 10 v/v%.
Preferably, the basic medium is prepared by the following method:
DMEM powder 13.4g, NaHCO33.7g of HEPES powder, 3.57g of penicillin, 100mg of streptomycin and Mill-Q ultrapure water are subjected to constant volume to 1L, the pH value is 7.0-7.4, the osmotic pressure is 280-320 mOsm/kg, and a 0.22 mu m filter membrane is used for filtering and sterilizing to obtain the basal culture medium.
Preferably, in the step (2), the neutralizing comprises the steps of:
and mixing the neutralization solution with the digestive juice for neutralization.
Preferably, in the step (2), the volume ratio of the neutralization solution to the digestive juice is 0.5-1.5:0.5-1.5, preferably 0.8-1.2:0.8-1.2, and more preferably 1: 1.
Preferably, the filtration pore size is 50-150 μm, preferably 80-120 μm, more preferably 100 μm.
Preferably, the filtration is performed by using a stainless steel filter screen.
Preferably, in the step (3), the centrifugation time is 2-8min, preferably 4-6min, and more preferably 5 min.
Preferably, in the step (3), the rotation speed of the centrifugation is 1000-.
Preferably, in the step (3), the sediment is preadipocytes.
Preferably, in the step (3), the obtained preadipocytes are resuspended through basal culture with FBS to obtain a preadipocyte resuspension solution.
Preferably, the basal medium comprises DMEM basal medium.
Preferably, in the basic medium added with FBS, the content of the FBS is 5-15 v/v%, preferably 8-12 v/v%, and more preferably 10 v/v%.
Preferably, the basic medium is prepared by the following method:
DMEM powder 13.4g, NaHCO33.7g of HEPES powder, 3.57g of penicillin, 100mg of streptomycin and Mill-Q ultrapure water are subjected to constant volume to 1L, the pH value is 7.0-7.4, the osmotic pressure is 280-320 mOsm/kg, and a 0.22 mu m filter membrane is used for filtering and sterilizing to obtain the basal culture medium.
In a second aspect, the present invention provides a preadipocyte prepared by the method of the first aspect of the invention.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments.
Drawings
FIG. 1 is a graph of bovine intramuscular preadipocytes and growth curves.
FIG. 2 shows the microscopic examination of intramuscular preadipocytes of cattle.
FIG. 3 is a morphological observation of in vitro culture of bovine intramuscular preadipocytes, wherein (a) 24h of bovine intramuscular preadipocytes were inoculated 100 ×; (b) culturing bovine intramuscular preadipocytes for 48h in vitro by 200 x; (c 100X, d 200X, e 200X, f 100X) is insulin 10. mu.g/mL + dexamethasone 0.25. mu. mol/L before inducing differentiation to the 6d, 2d, 6d, 6d, oil red O staining, respectively (arrow indicates oil red O stained fat drop).
Fig. 4 shows that the difference of the letters a, b, c and d in the fat content during the differentiation of bovine intramuscular preadipocytes indicates significant difference (P < 0.05).
Detailed Description
The invention discloses a preparation method of preadipocytes, which comprises the steps of carrying out incubation digestion on fat by using a collagenase I solution, neutralizing a digestion solution obtained, filtering, centrifuging a filtrate obtained, and obtaining preadipocyte sediment. The preadipocytes prepared by the method have strong activity, the growth curve of in vitro culture is similar to S type, the preadipocytes conform to the normal growth and proliferation rule of in vitro culture cells, the ploidy of chromosomes is normal after continuous passage, the basic genetic characteristics of normal cells are kept, and the preadipocytes have excellent in vitro adipogenic capacity after induced differentiation.
Term(s) for
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.
As used herein, the terms "comprising," "including," and "containing" are used interchangeably and include not only open-ended definitions, but also semi-closed and closed-ended definitions. In other words, the term includes "consisting of … …", "consisting essentially of … …".
As used herein, the term "FBS" is fetal bovine serum.
As used herein, the term "DPBS" refers to a duchenne phosphate buffer.
Preparation method of preadipocytes
The invention provides a preparation method of preadipocytes, which comprises the following steps:
(1) mixing fat and a collagenase I solution, and then carrying out incubation digestion to obtain a digestive juice;
(2) neutralizing the digestive juice with a neutralizing solution, and filtering to obtain a filtrate;
(3) and centrifuging the filtrate, and collecting the precipitate to obtain the preadipocytes.
In a preferred embodiment of the present invention, the preadipocytes are selected from the group consisting of: intramuscular preadipocytes, subcutaneous preadipocytes, visceral preadipocytes, or a combination thereof.
In a preferred embodiment of the present invention, in the step (1), the fat is derived from a human or non-human mammal.
In a preferred embodiment of the present invention, the non-human mammal is a cow, sheep, dog, pig, horse, rabbit or cat.
In a preferred embodiment of the present invention, in the step (1), the fat is selected from the group consisting of: intramuscular fat, subcutaneous fat, visceral fat, or a combination thereof.
In a preferred embodiment of the present invention, in the step (1), the fat includes a crushed (or cut) fat.
In a preferred embodiment of the invention, the comminuted fat comprises fat pieces.
In a preferred embodiment of the present invention, the fat mass has a size of 0.2 to 2mm3Preferably 0.5-1.5mm3Preferably 0.8-1.2mm3Preferably 1mm3。
In a preferred embodiment of the present invention, in the step (1), the weight ratio of the fat to the collagenase type I solution is 0.5-1.5:0.5-1.5, preferably 0.8-1.2:0.8-1.2, more preferably 1: 1.
In a preferred embodiment of the present invention, in the step (1), the collagenase type I is contained in the collagenase type I solution in an amount of 0.5-1mg/mL, preferably 0.8mg/mL, and more preferably 1 mg/mL.
In a preferred embodiment of the present invention, in the step (1), the collagenase type i solution is prepared by the following method:
weighing type I collagenase, dissolving the type I collagenase in DPBS, and adjusting the pH value to 7.2-7.4 to obtain a type I collagenase solution.
Typically, in step (1), the collagenase type i solution is prepared by the following method:
weighing collagenase type I, dissolving the collagenase type I in DPBS, adjusting the pH value to 7.2-7.4, and filtering to obtain collagenase type I solution.
In a preferred embodiment of the present invention, the filtration is a 0.22 μm membrane filtration.
In a preferred embodiment of the present invention, in the step (1), the fat comprises washed fat.
In a preferred embodiment of the present invention, the washed fat comprises a buffer-washed fat.
In a preferred embodiment of the present invention, the buffer comprises a DPBS buffer solution.
In a preferred embodiment of the present invention, in the step (1), the temperature of the incubation digestion is 20-40 ℃, preferably 30-37 ℃, and more preferably 37 ℃.
In a preferred embodiment of the present invention, in the step (1), the incubation digestion time is 0.5-1.5h, preferably 0.8-1.2h, and more preferably 1 h.
In a preferred embodiment of the present invention, in the step (1), the mixture is shaken every 4-6min for 1 time during the incubation digestion.
In a preferred embodiment of the present invention, in the step (2), the neutralization solution comprises a basal medium.
In a preferred embodiment of the present invention, in the step (2), the neutralizing solution includes a basic medium added with FBS.
Preferably, the basal medium comprises DMEM basal medium.
Preferably, in the basic medium added with FBS, the content of the FBS is 5-15 v/v%, preferably 8-12 v/v%, and more preferably 10 v/v%.
Typically, the basal medium is prepared by the following method:
DMEM powder 13.4g, NaHCO33.7g of HEPES powder, 3.57g of penicillin, 100mg of streptomycin and Mill-Q ultrapure water are subjected to constant volume to 1L, the pH value is 7.0-7.4, the osmotic pressure is 280-320 mOsm/kg, and a 0.22 mu m filter membrane is used for filtering and sterilizing to obtain the basal culture medium.
Preferably, in the step (2), the neutralizing comprises the steps of:
and mixing the neutralization solution with the digestive juice for neutralization.
In a preferred embodiment of the present invention, in the step (2), the volume ratio of the neutralization solution to the digestion solution is 0.5-1.5:0.5-1.5, preferably 0.8-1.2:0.8-1.2, and more preferably 1: 1.
In a preferred embodiment of the invention, the filtration pore size is 50-150. mu.m, preferably 80-120. mu.m, more preferably 100. mu.m.
Preferably, the filtration is performed by using a stainless steel filter screen.
In a preferred embodiment of the present invention, in the step (3), the centrifugation time is 2-8min, preferably 4-6min, and more preferably 5 min.
In a preferred embodiment of the present invention, in the step (3), the rotation speed of the centrifugation is 1000-.
In a preferred embodiment of the present invention, in the step (3), the pellet is preadipocytes.
In a preferred embodiment of the present invention, in the step (3), the obtained preadipocytes are resuspended in a basal culture supplemented with FBS, so as to obtain a preadipocyte resuspension solution.
Preferably, the basal medium comprises DMEM basal medium.
Preferably, in the basic medium added with FBS, the content of the FBS is 5-15 v/v%, preferably 8-12 v/v%, and more preferably 10 v/v%.
Preferably, the basic medium is prepared by the following method:
DMEM powder 13.4g, NaHCO33.7g of HEPES powder, 3.57g of penicillin, 100mg of streptomycin and Mill-Q ultrapure water are subjected to constant volume to 1L, the pH value is 7.0-7.4, the osmotic pressure is 280-320 mOsm/kg, and a 0.22 mu m filter membrane is used for filtering and sterilizing to obtain the basal culture medium.
The main excellent technical effects of the invention comprise:
the preadipocytes prepared by the preparation method of the preadipocytes have strong activity, the growth curve of in vitro culture is similar to an S shape, the normal growth and proliferation rule of the in vitro culture cells is met, the ploidy of chromosomes is normal after continuous passage, the basic genetic characteristics of normal cells are kept, and the preadipocytes have excellent in vitro adipogenic capacity after induced differentiation.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Example 1 isolation and culture of bovine preadipocytes in vitro and characterization thereof
Type i collagenase digest: weighing 100mg of collagenase type I, dissolving in 100mL of DPBS, adjusting the pH value to 7.2-7.4, filtering and sterilizing by using a 0.22 mu m filter membrane, and storing at the temperature of-20 ℃.
DMEM basal medium: DMEM powder 13.4g, NaHCO33.7g, 3.57g of HEPES powder, 66mg of penicillin, 100mg of streptomycin and Mill-Q ultrapure water are subjected to constant volume to 1L, the pH value is 7.0-7.4, the osmotic pressure is 280-320 mOsm/kg, and the mixture is filtered and sterilized by a 0.22 mu m filter membrane and stored at the temperature of 4 ℃. Cell culture was used with 10% FBS (v/v).
1. In vitro isolation culture of bovine preadipocytes
The method for in vitro isolation and culture of bovine preadipocytes comprises the following steps:
(1) taking white fat between muscle bundles of cattle, removing macroscopic fiber components, muscle and blood vessel on the surface of adipose tissue block with ophthalmology scissors and tweezers, and cutting to obtain adipose tissue block with diameter of about 1mm3Size, transfer to 50mL centrifuge tube.
(2) Washing the sheared adipose tissue with DPBS buffer solution for several times, adding collagenase I digestive juice with the same weight as the sheared adipose tissue, and digesting in a constant-temperature water bath kettle at 37 deg.C for 1h (shaking for 1 time every 5 min) to obtain digestive juice; then, the digestion solution was neutralized by mixing with an equal volume of DMEM basal medium (containing 10 v/v% FBS), and then filtered through a sterile stainless steel filter sieve having a pore size of 100 μm, and the filtrate was collected.
(3) And (3) centrifuging the collected filtrate in another clean sterile centrifuge tube for 5min (1200rpm), removing floating fat cells and supernatant to obtain a precipitate which is bovine intramuscular preadipocyte precipitate, and adding a DMEM (DMEM) basic culture medium (containing 10 v/v% FBS) to resuspend the precipitated cells to obtain the bovine intramuscular preadipocyte resuspension solution.
2. Living cell assay
By 5X 104Per cm2Inoculating bovine intramuscular preadipocytes into a 6-well culture plate at a density of (1) and placing the culture plate in CO2Incubator (37 ℃, saturated humidity, 5% CO)2) Medium culture; changing the solution after culturing for 24h to remove cells which are not attached to the wall, and then changing the solution for 1 time every 2-3 d; when the separated cells grow to confluence (70% -80%), adding trypsin to digest the adherent cells; after the adherent cells are digested, collecting the cells in a 15mL centrifuge tube, centrifuging at 1200rpm for 5min, and discarding the supernatant; resuspend the cell pellet by adding 4 ℃ precooled cryo-fluid (DMEM + 20% FBS + 10% DMSO) to a cell concentration of about 107Transferring the cells to a freezing tube per mL; pre-cooling in a refrigerator at 4 deg.C for 30min, and quickly freezing the tube with cells in liquid nitrogen.
The activity of the cells was detected using trypan blue exclusion assay.
3. Cell growth viability assay
When the cultured bovine intramuscular preadipocytes are continuously passaged to the 3 rd time, the cells to be cultured reach a confluent state (70% -80%), are equivalently inoculated to a 24-hole culture plate, are randomly divided into 8 groups, each group has 3 holes, the total number of the cells in each hole in one group is detected every 2 days (d), the average value of the 3 holes is taken, and the cell growth activity detection result is shown in figure 1 after the 8 th group is finished:
as can be seen from figure 1, the growth curve of the bovine intramuscular preadipocytes is similar to S-shaped, and accords with the normal growth and proliferation rule of in vitro cultured cells, namely, the growth enters a slow growth latent period (about 40h), then reaches an exponential growth period (4 th to 10 th days), and finally reaches a growth stop plateau period (after 10 th days), thereby indicating that the bovine intramuscular preadipocytes have excellent activity.
4. The detection steps of the chromosome karyotype of the cells cultured in vitro are as follows:
when the cultured bovine intramuscular preadipocytes enter a logarithmic growth phase, adding colchicine solution (the final concentration is 0.06-0.1 mu g/mL) into an incubator for incubation for about 1 h; after the incubation is finished, digesting and collecting cells in a centrifuge tube by using trypsin digestive juice; centrifuging at 1200rpm for 10min, and discarding the supernatant; adding 5mL of preheated KCl hypotonic solution into the mixture, and performing water bath at 37 ℃ for 20 min; after hypotonic reaction, pre-fixing with 0.5mL of fixing solution; 1200rpm, centrifuging for 10min, collecting the precipitate, and fixing for 20min at the room temperature by using 5mL of stationary liquid; centrifuging at 1200rpm for 10min, and discarding the supernatant; carrying out secondary fixation for 15min at the room temperature by using 5mL of a fixation solution; centrifuging at 1200rpm for 10min, and discarding the supernatant; carrying out third fixation for 15min at the room temperature by using 5mL of a fixation solution; centrifuging at 1200rpm for 10min, and resuspending the collected precipitate with appropriate amount of fixing solution; dripping a few drops of cell suspension on a wet and cold glass slide, and fixing by using alcohol lamp flame; standing at room temperature, and air drying; after the chromosome specimen is dried in the air, performing knot dyeing for 10min at room temperature by using 10% Giemsa solution, washing with running water, and then performing air drying at room temperature; the microscopic examination result of intramuscular preadipocytes of cattle is shown in FIG. 2
As can be seen from FIG. 2, after the bovine intramuscular preadipocytes are cultured in vitro for continuous passage, the chromosome karyotype analysis is carried out to identify that the chromosome ploidy is still normal, the diploid cells in the in vitro culture state occupy obvious advantages, and the basic genetic characteristics of normal cells in an animal body are retained.
5. Isolated preadipocyte adipogenic Capacity assay
When the bovine intramuscular preadipocytes grow for 3-5 days in the basal medium to reach the cell confluent state, the bovine intramuscular preadipocytes are changed into a differentiation medium to induce differentiation culture for 10 days, and cells with 2d, 4d, 6d, 8d and 10d of induction are collected respectively to detect the accumulation amount of fat in cytoplasm. The intracellular fat content was determined for 2-10 days according to the method of Ramirez-Zaciaias et al (1992). After fixing the adherent cells of the culture plate for 1h by using an isotonic salt buffer solution containing 10% formaldehyde, rinsing with distilled water. 2mL of oil red O working solution is sucked to dip-dye the culture plate, the oil red O working solution is poured out after 2h, and the culture plate is rinsed for a plurality of times by distilled water until the culture plate is completely rinsed. The stained plate was placed in an oven at 32 ℃ to evaporate water from all wells of the plate, followed by addition of 1.5mL of isopropanol to extract for 20min, after which the staining solution was removed and the absorbance measured at a wavelength of 510nm in a spectrophotometer. Wherein, a part of the fat cells are observed under an inverted phase contrast microscope after being dyed, and the picture is photographed, and an oil red O dyeing picture is collected, wherein the oil red O dyeing shows that the morphological observation result of the bovine intramuscular preadipocytes is shown in figure 3, and the change of the fat content in the differentiation process of the bovine intramuscular preadipocytes is shown in figure 4.
As can be seen from fig. 3 and 4, small lipid droplets begin to appear in the cytoplasm of the subcultured bovine intramuscular preadipocytes on the 2 nd day after the cells are induced to differentiate in the differentiation medium to a fused state, and by the 6 th day, a large number of small lipid droplets appear in the cytoplasm of the cells, and a part of small lipid droplets begin to gradually fuse into large lipid droplets, indicating that the isolated bovine intramuscular preadipocytes have excellent in vitro adipogenic capability.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims (10)

1. A method for preparing preadipocytes, said method comprising the steps of:
(1) mixing fat and a collagenase I solution, and then carrying out incubation digestion to obtain a digestive juice;
(2) neutralizing the digestive juice with a neutralizing solution, and filtering to obtain a filtrate;
(3) and centrifuging the filtrate, and collecting the precipitate to obtain the preadipocytes.
2. The method of claim 1, wherein in step (1), the weight ratio of fat to collagenase i solution is 0.5-1.5:0.5-1.5, preferably 0.8-1.2:0.8-1.2, more preferably 1: 1.
3. The method according to claim 1, wherein in step (1), collagenase type i is present in the collagenase type i solution in an amount of 0.5-1mg/mL, preferably 0.8mg/mL, more preferably 1 mg/mL.
4. The method of claim 1, wherein in step (1), the temperature of the incubation digestion is 20-40 ℃, preferably 30-37 ℃, and more preferably 37 ℃.
5. The method of claim 1, wherein in step (1), the incubation digestion time is 0.5-1.5h, preferably 0.8-1.2h, more preferably 1 h.
6. The method of claim 1, wherein in step (2), the neutralizing solution comprises a basal medium supplemented with FBS.
7. The method according to claim 6, wherein the content of FBS in the FBS-supplemented basal medium is 5-15 v/v%, preferably 8-12 v/v%, more preferably 10 v/v%.
8. The method according to claim 1, wherein in step (2), the volume ratio of the neutralization solution to the digestion solution is 0.5-1.5:0.5-1.5, preferably 0.8-1.2:0.8-1.2, more preferably 1: 1.
9. The method of claim 1, wherein in step (3), the preadipocytes obtained are resuspended in basal culture supplemented with FBS to obtain a preadipocyte resuspension solution.
10. A preadipocyte cell produced by the method of claim 1.
CN202110381028.4A 2021-04-09 2021-04-09 Separation method of preadipocytes Pending CN113174367A (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102140437A (en) * 2011-01-07 2011-08-03 西北农林科技大学 Culturing method for quickly dedifferentiating porcine mature fat cell into precursor fat cell
CN103060268A (en) * 2013-01-18 2013-04-24 青岛农业大学 Method for culturing goat precursor fat cells in vitro
CN105969724A (en) * 2016-06-08 2016-09-28 西北民族大学 Separating culturing method for pig precursor adipose cells
CN109182256A (en) * 2018-08-31 2019-01-11 浙江省海洋水产研究所 A kind of spotted maigre Preadipocyte In Vitro be separately cultured and its abductive approach
CN109628404A (en) * 2018-12-18 2019-04-16 浙江大学 The construction method and purposes of Preadipocyte immortalized cell line under pigskin
CN112920993A (en) * 2021-04-23 2021-06-08 扬州大学 High-purity duck intramuscular precursor fat cell separation method
CN113973868A (en) * 2021-10-09 2022-01-28 百色学院 Method for making oat and purple sweet potato cake

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102140437A (en) * 2011-01-07 2011-08-03 西北农林科技大学 Culturing method for quickly dedifferentiating porcine mature fat cell into precursor fat cell
CN103060268A (en) * 2013-01-18 2013-04-24 青岛农业大学 Method for culturing goat precursor fat cells in vitro
CN105969724A (en) * 2016-06-08 2016-09-28 西北民族大学 Separating culturing method for pig precursor adipose cells
CN109182256A (en) * 2018-08-31 2019-01-11 浙江省海洋水产研究所 A kind of spotted maigre Preadipocyte In Vitro be separately cultured and its abductive approach
CN109628404A (en) * 2018-12-18 2019-04-16 浙江大学 The construction method and purposes of Preadipocyte immortalized cell line under pigskin
CN112920993A (en) * 2021-04-23 2021-06-08 扬州大学 High-purity duck intramuscular precursor fat cell separation method
CN113973868A (en) * 2021-10-09 2022-01-28 百色学院 Method for making oat and purple sweet potato cake

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
万荣等: "成年鲁西牛肌内前脂肪细胞的分离培养", 《农业生物技术学报》 *

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