CN105969724A

CN105969724A - Separating culturing method for pig precursor adipose cells

Info

Publication number: CN105969724A
Application number: CN201610402660.1A
Authority: CN
Inventors: 张国华; 卢建雄; 陈妍; 臧洪伟
Original assignee: Northwest Minzu University
Current assignee: Northwest Minzu University
Priority date: 2016-06-08
Filing date: 2016-06-08
Publication date: 2016-09-28
Anticipated expiration: 2036-06-08
Also published as: CN105969724B

Abstract

The invention relates to the field of cell engineering and tissue, in particular to a separating culturing method for pig precursor adipose cells. According to the separating culturing method, the existing separating culturing method is improved; after I-type collagenase is digested, cells are treated with the cold digestion technology, the precursor adipose cells are fully separated in a mode that grinding beads are added, nylon sieve filtering operation, centrifugation operation and red blood cell pyrolysis operation are canceled, and therefore the technical effects that experimental time is prolonged to more thorough carry out the freedom degree and the precursor-adipose-cell separation degree, operation procedure is simplified, the pollution probability is decreased, and damage to the precursor adipose cells is reduced are achieved.

Description

A kind of isolated culture method of porcine preadipocyte

Technical field

The present invention relates to cell engineering and tissue areas, thin in particular to a boar precursor fatty The isolated culture method of born of the same parents.

Background technology

From fatty tissue, the cell of separation and Culture is referred to as " Preadipocyte In Vitro ".Preadipocyte In Vitro It is the class adipocyte precursor with proliferation and differentiation ability, polyester under the influence of many factors in vivo Break up and become mature fat cell.The In vitro culture of Preadipocyte In Vitro can not only intactly recognize fat Fat tissue occurs and the overall process of hypertrophy, and can directly observe the various factors tune to this process Control.Additionally, lipocyte proliferation and differentiation meeting out of control are causeed fat, and with sugar and lipid metabolism, machine The body energy balance, obesity, type ii diabetes etc. have very close relationship.Therefore, select properly Animal model reappear the loose overall process of Preadipocyte In Vitro propagation in vitro for inquiring into above-mentioned life Significant with lysis.

Pig is the animal that obese degree is the highest, and lipidosis is the rapidest, and with Rodents mammal and The lipidosis position of birds and depositional model Rodents mammal and the lipidosis position of birds and There is significant difference in depositional model, with people's more closely, thus the lipocyte proliferation of pig and differentiation Pattern provides an ideal model for research human body lipidosis, simultaneously for research different animals Lipidosis pattern provide reference.At present, domestic oneself be successfully established rat, mice, people, chess The Preadipocyte In Vitro Isolation and culture model of cattle and rabbit, but porcine preadipocyte cultivating system Set up ripe not enough, remain porcine preadipocyte complex operation at present, adipose cell divides From degree not, the problem such as cell vulnerable to pollution in separation process.

In view of this, the special proposition present invention.

Summary of the invention

It is an object of the invention to provide the isolated culture method of a kind of porcine preadipocyte, described Isolated culture method has that easy and simple to handle, adipose cell separation degree is high, cell is difficult in separation process The advantage such as it is contaminated.

In order to realize the above-mentioned purpose of the present invention, spy by the following technical solutions:

The isolated culture method of a kind of porcine preadipocyte, comprises the following steps:

1), separate 3～5 age in days piglet subcutaneus adipose tissues and remove in fatty tissue visible blood vessel and Connective tissue；Join together with grinding bead after rinsing and tissue being shredded and fill NTx enzymatic solution Container in carry out digestion and obtain Digestive system；

2) I type is removed by the method for stratification after adding cell culture fluid mixing after, digestion terminates Collagenase solution；Shaking described container makes grinding bead be inoculated into culture bottle after being ground by remaining tissue In cultivate；

3), cultivating until cell is to digest cell after 75%～85% and be inoculated in cultivation to degrees of fusion Plate is cultivated standby.

The propagation of adipose cell, differentiation deposit closely related with human obesity and poultry body fat transition, close In the hot issue that the over-deposit Chinese scholars especially of human obesity and body fat is paid close attention to.And fat Fat precursor cultivates the basis being typically these researchs.

Owing to containing more precursor fatty tissue in 3～5 age in days pig bodies, so selecting the son in this stage Pig is as laboratory animal.But the amount of the piglet fatty tissue in this stage is less, so before obtaining every time The amount of body fat cell is limited, and this method, in existing technical foundation, adds grinding bead shock group Knit the operation of block, by cell separation the most out can considerably increase obtaining of adipose cell Taken amount.

Additionally, digest is needed to filter (by 100 μm and 25 μm) through 2 nylon mesh by prior art Digestion product and 3 centrifugal treating, polluting probability increases.And the present invention remove nylon mesh filter and from Heart step, simplifies operating process, reduces pollution probability, also reduces operating process to carefully simultaneously The injury of born of the same parents.

Preferably, the isolated culture method of porcine preadipocyte as above, in step 1) in, Remove in fatty tissue and also include before visible blood vessel and connective tissue:

Piglet subcutaneus adipose tissue 8min～15min is soaked with containing dual anti-PBS.

Fatty tissue can be softened with soaking containing dual anti-PBS, and make operation get a clear view, easily In the operation that blood vessel and connective tissue are peeled off.

Preferably, the isolated culture method of porcine preadipocyte as above, in step 1) in, Described rinsing is specially and rinses 2～3 times with containing dual anti-PBS.

Dual anti-Penicillium digitatum element and streptomycin.

Preferably, the isolated culture method of porcine preadipocyte as above, described by tissue shear Tissue is cut into 0.5mm by broken being specially³～1.5mm³Fritter.

Preferably, the isolated culture method of porcine preadipocyte as above, described grinding bead is One or more in zirconium oxide bead, steel ball, bead, ceramic bead.

As long as the selection of grinding bead meets the pearl of the characteristics such as high intensity, high-wearing feature and low oil suction ink rate Son, such as alumina lap pearl, Zirconium orthosilicate. pearl etc..

Preferably, the isolated culture method of porcine preadipocyte as above, in step 1) in, In described NTx enzymatic solution, the mass percent of NTx enzyme is 0.08%～0.12%；I type glue The volume of protoenzyme solution be shred after tissue 2～4 times；

The condition of described digestion is:

2 DEG C～6 DEG C digestion 10h～16h, subsequently 36～38 DEG C of digestion 15min～30min.

The chemistry entitled collagen hydrolysate enzyme (Collagenase) of collagenase, it can be in physiology PH and temperature Specifically hydrolyze the three-dimensional spiral structure of natural collagen protein under the conditions of degree, and do not damage other albumen Matter and tissue.NTx enzyme is used for epithelium, lung, fat and the separation of adrenal tissue's cell, because of This is applicable to the present invention.

After prior art sampling, it is directly placed into 37 DEG C of digestion 60min～90min, from cell separation to carefully Born of the same parents inoculate, and staff's spent time is long, sometimes due to the arrangement of time, it is necessary to stay up late；And this Method uses cold digestion method, can put into refrigerator and carry out after adding the NTx enzyme of 0.08%～0.12% Digested overnight, places into 36 DEG C～38 DEG C digestion 15min～30min on the 2nd day, and staff can basis Arrangement of time situation, selects cold digestion or heat digestion, improves selection space and the degree of freedom of experiment. Additionally, digestion the most excessively digestion under the conditions of 37 DEG C, cause cell attachment, growth the best, cold digestion This can be improved.

Preferably, the isolated culture method of porcine preadipocyte as above, in step 2) in, The method of described stratification particularly as follows:

A), digestion terminate after, in described container add with the isopyknic cell of described Digestive system cultivate Liquid, abandons supernatant after standing 8min～12min；

B), add 10ml～20ml cell culture fluid, shake up cleaning digest gently, stand Supernatant is abandoned after 8min～12min；

C), step B twice is repeated.

Digestion is typically carried out in the conical flask of 50ml, and it is molten that the method for stratification removes NTx enzyme Liquid, prevents digesting excessive damage cell and collagenase remaining causes cell attachment difficulty.

In step B) in, the when of cleaning, action wants light, and in this time, Preadipocyte In Vitro does not also have Separating, the purpose of cleaning is to preferably remove the digestive enzyme outside tissue.

Preferably, the isolated culture method of porcine preadipocyte as above, in step 2) in, Shake described container grinding bead to be inoculated in culture bottle after being ground by remaining tissue carry out cultivating Operation specifically includes:

After abandoning supernatant, the most firmly shake described container, make described grinding bead clash into digest, shake Add 10ml～20ml cell culture fluid after 1min～3min, continue to shake 1min～3min, by upper liquid Body is inoculated in culture bottle.

After digestion process above, it is the laxest that tissue has digested.Firmly shaking of this step Dynamic, under the mechanical force percussion grinding pearl, Preadipocyte In Vitro just sheds from small tissue blocks Come.Need subsequently to pour in culture bottle by supernatant liquid rapidly, note trying not piece of tissue to fall Enter.

Preferably, the isolated culture method of porcine preadipocyte as above, step 3) operation Particularly as follows:

Change liquid, cleaning after cell inoculation 2～3h, continue to cultivate；Treat that cell cultivation to degrees of fusion is Outwell culture medium after 75%～85%, clean with the tryptic digestive juice of PBS and 0.2%～0.3% successively Cell；

The tryptic digestive juice adding 0.2%～0.3% hatches to the cell rounding of 85%～95% stopping Digestion；

Cleaning and blow and beat cell with cell culture fluid makes cell come off from the bottom of culture bottle bottle, is inoculated in cultivation Plate is cultivated standby.

According to the feature that erythrocyte is the most adherent, change liquid after 2～3h and can effectively remove erythrocyte.Existing skill In art, it will usually use and add erythrocyte cracked liquid and coordinate centrifugal method removal erythrocyte, but this Operation can increase the operation of operation, it is often more important that erythrocyte cracked liquid may be to Preadipocyte In Vitro Cause extra damage.The application is with changing liquid behind 2～3h, and the operation cleaning 1～2 time can reach equally Removing erythrocytic effect, condition milder, the damage to Preadipocyte In Vitro is less.

The process of renewed vaccination is also a process of purifying cells, although having changed liquid behind 2～3h Through eliminating most sarcoplast and erythrocyte, but the substrate being likely present other a small amount of is thin Born of the same parents, Preadipocyte In Vitro accounts for major part；Through renewed vaccination, a small amount of heteroproteose cell growth is not preponderated, Just can well be eliminated.

Wherein, the percent of tryptic digestive juice is quality percentage by volume, the pancreas egg of 0.2%～0.3% White enzymic digestion liquid i.e. concentration 0.2g/100ml～0.3g/100ml.

Preferably, the isolated culture method of porcine preadipocyte as above, described cell is cultivated Liquid is the DMEM/F12 culture medium containing the FBS that percentage by volume is 8%～12%.

Compared with prior art, the invention have the benefit that

1), prior art sampling after, be directly placed into 37 DEG C digestion 60min～90min, from cell separation Inoculating to cell, staff's spent time is long, sometimes due to the arrangement of time, it is necessary to stay up late； And this method uses cold digestion method, refrigerator can be put into after adding the NTx enzyme of 0.08%～0.12% Carrying out digested overnight, within the 2nd day, place into 36 DEG C～38 DEG C digestion 15min～30min, staff is permissible According to arrangement of time situation, select cold digestion or heat digestion, improve selection space and the freedom of experiment Degree.Additionally, digestion the most excessively digestion under the conditions of 37 DEG C, cause cell attachment, growth the best, cold This can be improved by digestion.

2), due to 3～5 age in days pigs, the amount of fatty tissue is less, so it is thin to obtain precursor fatty every time The amount of born of the same parents is limited, and this method, in existing technical foundation, has added grinding bead impinging tissue block, permissible By cell separation the most out.

3), in prior art, digest needs to filter (by 100 μm and 25 μm) through 2 nylon mesh, Pollution probability increases.And the present invention removes nylon mesh and filters, is centrifuged and erythrocyte splitting step, simplify Operating process, also reduces pollution probability, reduces the damage causing Preadipocyte In Vitro.

Accompanying drawing explanation

In order to be illustrated more clearly that the specific embodiment of the invention or technical scheme of the prior art, under The accompanying drawing used required in detailed description of the invention or description of the prior art will be briefly described by face, It should be evident that the accompanying drawing in describing below is some embodiments of the present invention, general for this area From the point of view of logical technical staff, on the premise of not paying creative work, it is also possible to obtain according to these accompanying drawings Obtain other accompanying drawing.

Fig. 1 is the cell growth after the cell cultivation of prior art and the embodiment of the present application 5 isolated Curve；

Fig. 2 is the cultivation photo after the cell inoculated and cultured plate 24h of embodiment 5 isolated；

Fig. 3 is the cell inoculated and cultured plate of embodiment 5 isolated cultivation photo after 5 days；

Fig. 4 is that the cell of embodiment 5 isolated is divided into mature fat cell, with oil after induction Red O dyes, and fat drips the photo after dying redness.

Detailed description of the invention

Below in conjunction with embodiment, embodiment of the present invention are described in detail, but this area skill Art personnel are it will be appreciated that the following example is merely to illustrate the present invention, and are not construed as limiting the present invention Scope.Unreceipted actual conditions person in embodiment, the condition advised according to normal condition or manufacturer Carry out.Agents useful for same or instrument unreceipted production firm person, being can be by commercially available purchase acquisition Conventional products.

Embodiment 1

Embodiment 2

1), separate 3～5 age in days piglet subcutaneus adipose tissues, soak son with containing dual anti-PBS Pig subcutaneus adipose tissue 8 minutes, removes in fatty tissue visible blood vessel and connective tissue and with containing pair Anti-PBS rinses 3 times；Tissue shear is also broken into 0.5mm by rinsing³～1.5mm³Fritter after The NTx enzymatic solution filling mass percent 0.08% is joined together with zirconium oxide bead, ceramic bead Container digests, prior to 2 DEG C of digestion 16h, 36 DEG C of digestion 30min subsequently.Wherein, I type glue The volume of protoenzyme solution be shred after tissue 2 times；

2), digestion terminate after, in described container add with the isopyknic cell of described Digestive system cultivate Liquid, abandons supernatant after standing 8min；Add 10ml cell culture fluid, shake up cleaning digest gently, Abandon supernatant after standing 8min, repeat this operation 2 times；

Uniformly firmly shake described container subsequently, make described grinding bead clash into digest, add after shaking 3min Enter 10ml cell culture fluid, continue to shake 1min, supernatant liquid is inoculated in culture bottle；

Described cell culture fluid is the DMEM/F12 culture medium containing the FBS that percentage by volume is 8%；

3), cultivating until cell is to digest cell after 85% and be inoculated in culture plate to degrees of fusion Cultivate standby.

Embodiment 3

1), separate 3～5 age in days piglet subcutaneus adipose tissues, soak son with containing dual anti-PBS Pig subcutaneus adipose tissue 15 minutes, removes in fatty tissue visible blood vessel and connective tissue and with containing pair Anti-PBS rinses 2 times；Tissue shear is also broken into 0.5mm by rinsing³～1.5mm³Fritter after Join in the container of the NTx enzymatic solution filling mass percent 0.12% to enter together with grinding bead Row digestion, prior to 6 DEG C of digestion 10h, 38 DEG C of digestion 15min subsequently.Wherein, NTx enzymatic solution Volume be shred after tissue 4 times；

2), digestion terminate after, in described container add with the isopyknic cell of described Digestive system cultivate Liquid, abandons supernatant after standing 12min；Add 20ml cell culture fluid, shake up cleaning digest gently, Abandon supernatant after standing 12min, repeat this operation 2 times；

Uniformly firmly shake described container subsequently, make described ceramic bead clash into digest, add after shaking 1min Enter 20ml cell culture fluid, continue to shake 3min, supernatant liquid is inoculated in culture bottle；

3), liquid, cleaning, continuation cultivation are changed after cell inoculation 2h；Treat that cell cultivation is 75% to degrees of fusion After outwell culture medium, clean cell with the tryptic digestive juice of PBS and 0.3% successively；

The tryptic digestive juice adding 0.3% hatches to the cell rounding of 95% stopping digestion；

Cleaning and blow and beat cell with cell culture fluid makes cell come off from the bottom of culture bottle bottle, is inoculated in cultivation Plate is cultivated standby；

Described cell culture fluid is that the DMEM/F12 containing the FBS that percentage by volume is 12% cultivates Base.

Embodiment 4

1), separate 3～5 age in days piglet subcutaneus adipose tissues, soak son with containing dual anti-PBS Pig subcutaneus adipose tissue 10 minutes, removes in fatty tissue visible blood vessel and connective tissue and with containing pair Anti-PBS rinses 3 times；Tissue shear is also broken into 0.5mm by rinsing³～1.5mm³Fritter after Join together with steel ball in the container of the NTx enzymatic solution filling mass percent 0.1% and disappear Change, prior to 4 DEG C of digestion 14h, 37 DEG C of digestion 15min subsequently.Wherein, the body of NTx enzymatic solution Amass as after shredding 4 times of tissue；

2), digestion terminate after, in described container add with the isopyknic cell of described Digestive system cultivate Liquid, abandons supernatant after standing 10min；Add 15ml cell culture fluid, shake up cleaning digest gently, Abandon supernatant after standing 10min, repeat this operation 2 times；

Uniformly firmly shake described container subsequently, make described grinding bead clash into digest, add after shaking 2min Enter 20ml cell culture fluid, continue to shake 3min, supernatant liquid is inoculated in culture bottle；

3), liquid, cleaning, continuation cultivation are changed after cell inoculation 3h；Treat that cell cultivation is 85% to degrees of fusion After outwell culture medium, clean cell with the tryptic digestive juice of PBS and 0.2% successively；

The tryptic digestive juice adding 0.2% hatches to the cell rounding of 85% stopping digestion；

Described cell culture fluid is that the DMEM/F12 containing the FBS that percentage by volume is 10% cultivates Base.

Embodiment 5

1, the separation of Adipose Tissue and digestion

Separate 3～5 age in days piglet subcutaneus adipose tissues under aseptic condition, the fatty tissue side cut is existed In glass dish, soak 10min with containing dual anti-PBS, remove macroscopic connective tissue After blood vessel, clean 2 times with containing dual anti-PBS, fatty tissue is transferred to another and does In clean glass dish, cut with ophthalmologic operation and fatty tissue is cut into 1mm³The fritter of left and right, afterwards by fat Fat tissue fritter proceeds in the conical flask with cover of a 50ml (added with the little glass of 5～10 sterilizings in bottle Glass pearl), in conical flask, add the 0.1% NTx enzyme being about 3 times of volumes of piece of tissue, build lid, With sealed membrane, bottleneck is sealed, put into 4 DEG C of refrigerators and carry out digesting 12h, within the 2nd day, place into 37 DEG C of concussions Water-bath digestion 20min.

2, the inoculated and cultured of Preadipocyte In Vitro

After digestion terminates, in conical flask, add the DMEM/F12 culture medium of isopyknic 10%FBS, After standing 10min, abandon supernatant, add the DMEM/F12 culture medium of 15ml 10% hyclone, Shake up gently, clean digest, abandon supernatant after standing 10min, repeat to wash twice, after abandoning supernatant, Uniformly firmly shake conical flask, allow bead clash into digest, after shaking 2min, add 15ml 10% tire The DMEM/F12 culture medium of Ox blood serum, after continuing to shake 2min, is quickly inoculated into training by supernatant liquid Support in bottle.It is placed in 37 DEG C, 5%CO₂Incubator is cultivated.Change liquid after 2h, use serum-free DMEM/F12 Culture medium washes away the most adherent cell, continues to be placed in 37 DEG C, 5%CO₂Incubator is cultivated.

3, the purification of Preadipocyte In Vitro

After cell inoculation 2h, changing liquid, PBS washes cell 2 times, adds the DMEM/F12 of the FBS of 10% Continue to cultivate, after cell is cultivated and merged to 80%, outwell culture medium, with PBS 1 time, use Incubation cross 0.25% tryptic digestive juice clean cell 1 time, then rejoin 3ml 0.25% Tryptic digestive juice, hatch a moment, the cell of basis of microscopic observation to 90% all becomes round, and drops to Digestive system, adds the DMEM/F12 culture medium containing 10% hyclone and cleans 1 time, be subsequently adding 6ml The DMEM/F12 culture medium of 10% hyclone, at the bottom of elbow straw piping and druming bottle, all blows cell Fall, with 5 × 10⁴/cm²Density is inoculated in culture plate, i.e. can be used for follow-up study.

Experimental example

Take fatty tissue 10ml and be respectively adopted prior art and most preferred embodiment embodiment 5 (is i.e. schemed Improvement technology in 1) in method carry out digestion separate obtain Preadipocyte In Vitro, through trypan blue contaminate Color, analysis of accounts, select identical raw material, the pig precursor fatty of the method isolated of embodiment 5 Cell concentration is 8.7 × 10⁴Individual/ml, and prior art is only 5.6 × 10⁴Individual/ml, carrying after i.e. improving The amount of access method separation acquisition porcine preadipocyte is significantly higher than the amount of the obtained cell of prior art. Two kinds of cell growth curves the most S-type (Fig. 1).The cellular morphology of improved method acquisition and prior art Not having difference, after inoculated and cultured plate 24h, cell is the most adherent, in shuttle-type or irregular shape (Fig. 2), After inoculated and cultured plate 5 days, along with cell proliferation, start between cell to merge (Fig. 3).Through induction, The cell that embodiment 5 separates also can be divided into mature fat cell, and with oil red O stain, fat drips and can contaminate Become red (Fig. 4).

For sake of convenience, the present invention only gives the effect of most preferred embodiment 5 in experimental example, but real On border, in the range of the record of the application claim, this effect all can realize, the cell of embodiment 1～4 Growth curve compared with prior art there are no significant difference, and cellular morphology is the most roughly the same.

As can be seen here, the isolated culture method of the porcine preadipocyte provided through the present invention separates To porcine preadipocyte achieve improve experiment time carry out degree of freedom, Preadipocyte In Vitro is divided On the premise of the technique effects such as probability are polluted in and reduction higher from degree, also maintain normal growth State.

Note: the embodiment of prior art is:

Separate 3～5 age in days piglet subcutaneus adipose tissues, PBS 3 times under aseptic condition, remove meat After the visible connective tissue of eye and blood vessel, it is cut into 1mm with shears³The small tissue blocks of left and right, puts into cover In bottle, (every 5min shakes to add 0.1% NTx enzyme 37 DEG C digestion about 60min～90min Once), then with the double-layer nylon of 100 μm and 25 μm it is sieved through and filters digest, by filtrate 1500r/m Centrifugal 10min, abandons supernatant, adds erythrocyte cracked liquid, and uniformly, room temperature stands 10min in piping and druming, 1000r/m is centrifuged 5min again, abandons supernatant, adds serum-free medium, and piping and druming uniformly, can obtain Porcine preadipocyte, 5 × 10⁴Individual/cm²Density Preadipocyte In Vitro is inoculated in 6 holes cultivate In plate, it is placed in 37 DEG C, 5%CO₂Incubator is cultivated.

Last it is noted that various embodiments above is only in order to illustrate technical scheme, rather than It is limited；Although the present invention being described in detail with reference to foregoing embodiments, but this area Those of ordinary skill it is understood that it still can be to the technical scheme described in foregoing embodiments Modify, or the most some or all of technical characteristic is carried out equivalent；And these amendments Or replace, do not make the essence of appropriate technical solution depart from the model of various embodiments of the present invention technical scheme Enclose.

Claims

1. the isolated culture method of a porcine preadipocyte, it is characterised in that comprise the following steps:

2. the isolated culture method of porcine preadipocyte as claimed in claim 1, it is characterised in that In step 1) in, remove in fatty tissue and also include before visible blood vessel and connective tissue:

3. the isolated culture method of porcine preadipocyte as claimed in claim 1, it is characterised in that In step 1) in, described rinsing is specially and rinses 2～3 times with containing dual anti-PBS.

4. the isolated culture method of porcine preadipocyte as claimed in claim 1, it is characterised in that Tissue is specially cut into 0.5mm by described being shredded by tissue³～1.5mm³Fritter.

5. the isolated culture method of porcine preadipocyte as claimed in claim 1, it is characterised in that Described grinding bead is one or more in zirconium oxide bead, steel ball, bead, ceramic bead.

6. the isolated culture method of porcine preadipocyte as claimed in claim 1, it is characterised in that In step 1) in, in described NTx enzymatic solution, the mass percent of NTx enzyme is 0.08%～0.12%；The volume of NTx enzymatic solution be shred after tissue 2～4 times；

The condition of described digestion is:

7. the isolated culture method of porcine preadipocyte as claimed in claim 1, it is characterised in that In step 2) in, the method for described stratification particularly as follows:

C), step B twice is repeated.

8. the isolated culture method of porcine preadipocyte as claimed in claim 1, it is characterised in that In step 2) in, shake described container and make grinding bead be inoculated into culture bottle after being ground by remaining tissue In the operation that carries out cultivating specifically include:

9. the isolated culture method of porcine preadipocyte as claimed in claim 1, it is characterised in that Step 3) operation particularly as follows:

10. the isolated culture method of the porcine preadipocyte as described in any one of claim 1～9, It is characterized in that, described cell culture fluid is containing the FBS's that percentage by volume is 8%～12% DMEM/F12 culture medium.