CN102936580B - Method for culturing adipose cells in vitro - Google Patents
Method for culturing adipose cells in vitro Download PDFInfo
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- CN102936580B CN102936580B CN201210454794.XA CN201210454794A CN102936580B CN 102936580 B CN102936580 B CN 102936580B CN 201210454794 A CN201210454794 A CN 201210454794A CN 102936580 B CN102936580 B CN 102936580B
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Abstract
The invention discloses a method for culturing adipose cells in vitro. The method includes: firstly treating adipose tissues into chyle shape in homogenate mode, then evenly coating the adipose tissues on the bottom wall of a culture bottle to form a sheet through an improved inoculating loop, adding culture liquid containing taurine by overturning the culture bottle, flatly placing the culture liquid into a culture box, overturning the culture bottle after 30-40 minutes, and enabling the culture liquid to enter the coated sheet on the bottom wall of the culture bottle for continuous culturing, wherein the cells can grow in adherence mode after 24-36 hours, and nearly 80-90% of the cells can adhere onto the wall in 4-5 days. Compared with a collagenase digestion method, the method is simple in operation procedures and short in spent time and generally causes no culture contamination, the cells are high in activity due to the fact that no influence from digestion of the collagenase can be caused, the adhered cells are good in growing status, and compared with a tissue block culture method, the cell culture period is greatly reduced, and the cells are even in growth.
Description
Technical field
The present invention relates to a kind of biological culture technigue, specifically a kind of adipocyte extracorporeal culturing method.
Background technology
Fatty tissue is energy bank maximum in body, and metabolism of fat is the most basic energy metabolism mode of life.Generally believe now that metabolism of fat can cause various diseases extremely, as obesity, atherosclerosis, type II diabetes, hypertension and blood lipid dysbolism etc.In order more in depth fat metabolic disturbance disease to be studied, investigator finds that setting up adipocyte in-vitro culture model is a kind of effective approach (Armani A, Mammi C, Marzolla V, et al..Cellular models for understanding adipogenesis, adipose dysfunction, and obesity.J Cell Biochem.2010,110 (3): 564-72.).
Adipocyte extracorporeal culturing method mainly contains two kinds at present, is respectively collagenase digestion and tissue mass cell culture.First collagenase digestion shreds fatty tissue, then add certain density collagenase, 37 ℃ of digestion 60-90 minute, every approximately 10 minutes, with suction pipe piping and druming, mix tissue during this time, afterwards the tissue juice of digestion is crossed to 200 order cell sieves, collected the centrifugal washed twice of filtering, finally add nutrient solution to put incubator and cultivate, cell attachment growth after about 16-18 hour, the 80-90% cell attachment of having an appointment after 3-4 days.The advantage of this method is that cell attachment is fast, growth evenly, yet complex steps when shortcoming is operation, spended time are longer, thereby easily cause to cultivate and pollute.Tissue mass cell culture is first fatty tissue to be shredded equally, then with suction pipe, tissue block is evenly emitted on to (approximately 3 millimeters, tissue block interval) on culturing bottle diapire, the culturing bottle that overturns afterwards adds nutrient solution, keep flat into incubator, after 2-3 hour, culturing bottle is overturn, allow nutrient solution immerse tissue block and continue to cultivate, after 96-108 hour, have cell from tissue block free out growth around, after about 10-12 days, have 80-90% cell attachment.The advantage of this method is that operation steps simply, generally can not cause cultivation to pollute, but shortcoming is also apparent, the one, and cell culture period is longer; The 2nd, Growth of Cells is inhomogeneous, and tissue block peripheral cell is grown due to too intensive and aging, but at this moment culturing bottle diapire also has segment space due to far away apart from tissue block, and cell is the not free adherent growth of coming also.
Summary of the invention
In order to overcome the shortcoming of above two kinds of adipocyte extracorporeal culturing methods, the invention provides a kind of extracorporeal culturing method of adipocyte.This cultural method is compared with collagenase digestion, and operation steps is simple, takes a short time, and conventionally can not cause to cultivate and pollute, and owing to not being subject to the impact of collagenase digesting, cytoactive is higher, and adherent rear growth conditions is good; Compare with tissue mass cell culture, cell culture period shortens greatly, and Growth of Cells is even.
Technical scheme of the present invention is: a kind of extracorporeal culturing method of adipocyte; it is characterized in that; the first homogenized of fatty tissue is become to chyle shape; then use transfering loop (preferably using improved transfering loop) that chyle is organized in to the even smear of culturing bottle diapire; the culturing bottle that overturns afterwards adds the nutrient solution containing taurine; keep flat into incubator; after 30-40 minute, culturing bottle is overturn; allowing nutrient solution enter in the smear of culturing bottle diapire continues to cultivate; cell attachment growth after 24-36 hour, the 80-90% cell attachment of having an appointment after 4-5 days.
Specifically comprise the following steps:
(1) homogenized of fatty tissue
First fatty tissue is placed on to plate in aseptic super clean bench in, be trimmed to 0.8-1.2cm
3bulk, then use phosphate buffered saline buffer (PBS) to rinse 3-5 time, afterwards tissue block is placed in dry plate and is stained with under 3-5, after making tissue block dry a little, put into the sterile test tube of tissue refiner, 180-200 rev/min of homogenized 3-5 minute, at this moment tissue block becomes chyle shape.
(2) adipocyte culture
With suction pipe, draw chyle tissue and be placed on culturing bottle diapire, then use improved transfering loop (between ring and handle, to become 30 degree angles, as shown in Figure 1) will be organized in even smear (thickness is 0.10-0.15 millimeter) on culturing bottle diapire, with transfering loop, shift residue chyle tissue onto bottle mouth position afterwards, suction pipe sucking-off discards; Then the culturing bottle that overturns adds the nutrient solution containing taurine, keeps flat in incubator, by culturing bottle upset gently again, allows nutrient solution slowly enter in the smear of culturing bottle diapire, and again put back to incubator and continue to cultivate after 30-40 minute.Cell attachment growth after 24-36 hour, the 80-90% cell attachment of having an appointment after 4-5 days.
(3) nutrient solution preparation and interpolation
By 50-60 part (volume parts) foetal calf serum (FBS), 2-3 part (volume parts) mycillin mixed solution, 0.1-0.2 part (mass fraction; the taurine that adds 0.1～0.2g in the cell culture fluid of every 450-500 milliliter) taurine is dissolved in 450-500 part (volume parts) cell culture fluid (DMEM/F12); after 0.22 micron of membrane filtration, divide and install in the centrifuge tube of sterilizing; every pipe 40-45 milliliter; place 4 ℃ of preservations, in two weeks, be finished.Before cell cultures, nutrient solution was placed on to preheating in 37 ℃ of water-baths in 0.5-1 hour.
In culturing bottle, add for the first time the amount of nutrient solution very crucial, should be in strict accordance with shown in table 1.
Table 1 nutrient solution addition table corresponding to culturing bottle model
The invention has the beneficial effects as follows: (1) the present invention first becomes chyle shape by fatty tissue homogenate, this processing not only than traditional free-hand shred save time, laborsaving, and make single tissue grain more even, tiny, after culturing bottle diapire smear tissue be easy to attached to it, so the time of culturing bottle upset is from the 30-40 minute of the present invention that shortens to for 2-3 hour of tissue mass cell culture.(2) the present invention is organized in culturing bottle diapire smear by chyle; thickness is 0.10-0.15 millimeter; almost being equivalent to cell monolayer is attached on diapire; cell is free growth easily; in nutrient solution, add again the taurine of promoting growth of cell; so the initial adherent growth time of cell shortens to 24-36 hour of the present invention in 96-108 hour from tissue mass cell culture, in culturing bottle, there is the time of 80-90% cell attachment to shorten to 4-5 days from 10-12 days, culture cycle shortens greatly.(3) the present invention compares with collagenase digestion, and during operation, step is simple, and spended time is shorter, conventionally can not cause to cultivate and pollute; And owing to not being subject to the impact of collagenase digesting and centrifuge washing, cytoactive is higher, adherent rear growth conditions is good, so the time that in the present invention, cell reaches the adherent degree of 80-90% was than collagenase digestion only late 1 day.(4) the present invention is organized in the even smear of culturing bottle diapire by chyle, and what from each tissue grain, cell free was grown has equal opportunities, so Growth of Cells is even.
Accompanying drawing explanation
Fig. 1 is the structural representation of improved transfering loop; In Fig. 1,1 is ring, and 2 is handle.
Fig. 2 is that subcutaneous fat cells adopts the method for embodiment 1 to cultivate 100 * photo after 3 days; Wherein arrow place is that after cell free growth, residual fat drips.
Embodiment
1, nutrient solution preparation
By 50 milliliters of FBS, 2.5 milliliters of mycillin mixed solution ［ penicillin-Streptomycin sulphate mixed solutions (100X); Penicillin Content is 10000U/ml; the content of Streptomycin sulphate is 10mg/ml ］, 0.15 gram of taurine is dissolved in 450 milliliters of DMEM/F12 nutrient solutions; after 0.22 micron of membrane filtration, divide and install in 50 milliliters of centrifuge tubes of sterilizing; 40 milliliters of every pipes, place 4 ℃ of preservations.Before cell cultures half an hour by two pipes totally 80 milliliters of nutrient solutions be placed on preheating in 37 ℃ of water-baths.
2, adipocyte culture
From the slaughterhouse clip subcutaneus adipose tissue inside Luxi Yellow cattle hind leg of growing up, be placed in the PBS liquid of 1% mycillin liquid 2 hours and take back laboratory.In aseptic super clean bench, first fatty tissue is placed in plate, be trimmed to 1 cubic centimetre of bulk, then with PBS liquid, rinse 5 times, during flushing, operator's left hand is held Cyphophthalmi tweezer and is clamped tissue block, the right hand is held Cyphophthalmi and is cut with its blunt end tissue block inside of repeatedly setting up a stall out, outside, afterwards tissue block is placed in dry plate and is stained with 3 times, then puts into the sterile test tube of tissue refiner after making tissue block dry a little, 200 revs/min of homogenized 5 minutes, at this moment tissue block becomes chyle shape.With suction pipe, draw chyle tissue and be placed on T75 culturing bottle diapire; then use improved transfering loop (as shown in Figure 1; between ring and handle, become 30 degree angles) will be organized on culturing bottle diapire evenly smear (thickness is 0.12 millimeter); with transfering loop, shift residue chyle tissue onto bottle mouth position afterwards; suction pipe sucking-off discards; the culturing bottle that finally overturns adds 15 milliliters containing the nutrient solution of taurine; keep flat in incubator; after 40 minutes, culturing bottle is overturn gently; allow nutrient solution slowly enter in the smear of culturing bottle diapire, and again put back to incubator and continue to cultivate.Cell attachment growth after 36 hours, the 50-60% cell attachment (as shown in Figure 2) of having an appointment after 3 days.
Claims (4)
1. an extracorporeal culturing method for adipocyte, is characterized in that,
(1) homogenized of fatty tissue
First fatty tissue is placed on to plate in aseptic super clean bench in, be trimmed to 0.8-1.2cm
3bulk, then with PBS damping fluid, rinse 3-5 time, afterwards tissue block is placed in dry plate and is stained with under 3-5, then put into sterile test tube 180-200 rev/min of homogenized 3-5 minute of tissue refiner, make tissue block become chyle shape;
(2) adipocyte culture
With suction pipe, draw chyle shape tissue and be placed on culturing bottle diapire, then with transfering loop, will be organized in even smear on culturing bottle diapire, plate coating thickness is 0.10-0.15 millimeter, shifts residue chyle shape tissue onto bottle mouth position, suction pipe sucking-off afterwards with transfering loop; Then the culturing bottle that overturns adds the nutrient solution containing taurine, keeps flat in incubator, after 30-40 minute, culturing bottle is overturn again, allows nutrient solution slowly enter in the smear of culturing bottle diapire, and again puts back to incubator and continue to cultivate.
2. the extracorporeal culturing method of adipocyte as claimed in claim 1; it is characterized in that; the described nutrient solution containing taurine is: by volume; 50-60 part foetal calf serum and 2-3 part mycillin mixed solution are dissolved in the cell culture fluid DMEM/F12 of 450-500 part, in every 450-500ml cell culture fluid DMEM/F12, add in addition the taurine of 0.1～0.2g simultaneously.
3. the extracorporeal culturing method of adipocyte as claimed in claim 1, is characterized in that, is 30 degree angles between the ring of described transfering loop and handle.
4. the extracorporeal culturing method of adipocyte as claimed in claim 1, is characterized in that, in described culturing bottle, the addition of nutrient solution is: in T25 type culturing bottle, add 4-5ml, add 12-15ml in T75 type culturing bottle.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1778905A (en) * | 2004-11-22 | 2006-05-31 | 赵春华 | Separating culture and use for fatty mesenchymal dry cell |
| 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 |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1778905A (en) * | 2004-11-22 | 2006-05-31 | 赵春华 | Separating culture and use for fatty mesenchymal dry cell |
| 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 |
Non-Patent Citations (4)
| Title |
|---|
| 刘晓牧等.去乙酰化酶1基因对牛前体脂肪细胞凋亡影响的研究.《生物化学与生物物理进展》.2010,第37卷(第3期), |
| 去乙酰化酶1基因对牛前体脂肪细胞凋亡影响的研究;刘晓牧等;《生物化学与生物物理进展》;20100315;第37卷(第3期);第70页左栏1.2培养方法 * |
| 大鼠成熟脂肪细胞原代培养方法比较;张艳等;《重庆医科大学学报》;20080115;第33卷(第1期);第298页左栏第3段1.2.1牛前体脂肪细胞培养方法 * |
| 张艳等.大鼠成熟脂肪细胞原代培养方法比较.《重庆医科大学学报》.2008,第33卷(第1期), |
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