CN101215547B

CN101215547B - Method for separating, purifying and extracting fat mesenchymal stem cell

Info

Publication number: CN101215547B
Application number: CN2008100011072A
Authority: CN
Inventors: 刘岱良
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-01-15
Filing date: 2008-01-15
Publication date: 2011-06-08
Anticipated expiration: 2028-01-15
Also published as: CN101215547A

Abstract

The invention relates to the biological cell extract and application field and provides a method for separating, purifying and extracting mesenchymal stem cell in the fat, which comprises the following steps: first, extracting fat source under sterile condition, soaking by buffer, rinsing, adopting nutrient solution to do water-bath oscillation and digestion, fetching the sieved digestion material to do centrifugal disposal, obtaining base microtubule layer, sedimenting and removing erythrocyte, using nutrient solution to centrifugal and rinse after sieving the digestion material, using physiological saline to dilute the digested and separated cell, separating electrophoresis tank into three parts transversely by nylon screen cloth evenly, placing potassium chloride and calcium gluconate solution according to certain bulk rate, inputting AC power for 2-5 min, placing the obtained cell in the middle electrophoresis tank to stir evenly, electrophorezing for 15-30 min under DC power, extracting cell liquid in the electrophoresis tank at the positive electrode side, obtaining the mesenchymal stem cell in the fat. The method is short in period without causing gene sorting disorder or mutation on the DNA vortex chain.

Description

The method of a kind of separation, purifying and extraction fat mesenchymal stem cell

Technical field

The present invention relates to biomass cells and extract and Application Areas, particularly relate to a kind of stem cell extracting method.

Background technology

Stem Cell Engineering is just paid attention to by more and more scholars as one of trend of future medicine.On October 8th, 2007, two U.S. scientists and the outstanding contributions of a Britain scientist aspect stem cell have been commended in Nobel's physiology or medical science prize.To the effect that utilize undifferentiated stem cell,, transform in vivo specific gene, and lay a good foundation for the development of " gene target " technology by homologous dna reorganization.This will produce a kind of brand-new medical skill, just screen human body gene, repair damaged tissue or organ by stem cell.

U.S. scientist finds, from the fat of patient posterior and thigh extraction, contains the cell of a large amount of similar stem cells, and these cells can develop into healthy cartilage and muscle etc.This discovery might make fat become the main source of stem cell, and scientist may will needn't extract stem cell from now on from embryonic tissue or marrow, thereby both reduces cost, and avoids arguement ethically again.

" natural science progress " the 17th volume report of publishing in February, 2007: " fat mesenchymal stem cell is that a class is present in the stem cell that has multidirectional differentiation potential in the fatty tissue.Studies have shown that in recent years: fatty tissue has draw materials convenient and the high advantage of stem cell content! Has more wide application prospect in research and Application Areas than bone marrow stem cell ".

In addition, stem cell also has outstanding advantage in anti-ageing field, and still, all these theoretical results will move towards practical clinical from the laboratory, also have suitable distance.Its major cause is that the stem cell extracting method is difficult for quick outputization, method that present stem cell extracting method mainly adopts is traditional " adherent, cultivate, go down to posterity ".The drawback of these class methods is that the cycle is oversize, and stem cell causes the gene that dna double revolves on the chain to sort disorderly or sudden change in the in-vitro multiplication reproduction process easily.

Summary of the invention

In view of above problem, the method that main purpose of the present invention is to provide a kind of separation, purifying and extracts fat mesenchymal stem cell.

The method of this kind separation provided by the invention, purifying and extraction fat mesenchymal stem cell mainly may further comprise the steps:

A, fat source extraction, separation, purifying

Fat source extracts under the aseptic condition, soak, wash by damping fluid then, adopt nutritive medium water-bath vibration digestion again, get filtered liquid after the gained digest sieves and carry out centrifugal treating, obtain its matrix microtubule layer, the precipitation part is removed red corpuscle, and then uses the nutritive medium centrifuge washing after the centrifugal treating, will digest isolated cell and dilute with physiological saline;

B, mescenchymal stem cell electrophoresis, extraction

Electrophoresis chamber evenly laterally is divided into three lattice with nylon wire screen, insert Repone K and calcium gluconate solution according to a certain volume, alternating-current continues input 2～5 minutes, the layout electrophoresis chamber stirs evenly in then above-mentioned gained cell solution being inserted, direct current electrophoresis 15～30 minutes, extract positive electrode side electrophoresis chamber inner cell liquid, promptly get fat mesenchymal stem cell.

Wherein, the swelling liquid preparation standard of the moist liposuction method employing in the above-mentioned steps one is preferably:

Physiological saline 1000ML;

2% lignocaine 20ML;

0.5% suprarenin 1ML.

The aperture of described nylon mesh and nylon wire screen all is preferably 50uM.

In particular, the method for this kind separation provided by the invention, purifying and extraction fat mesenchymal stem cell may further comprise the steps:

A, fat source extraction, separation, purifying

Moist liposuction method extracts fat under the aseptic condition, and adopt phosphate buffered saline buffer to soak, flushing, then with the serum-free DMEM nutritive medium water-bath vibration digestion that contains 0.1%I Collagen Type VI enzyme, the gained digest is got filtered liquid after sieving by nylon mesh, carry out centrifugal treating again, abandon supernatant and buoyant mature fat cell, obtain matrix microtubule layer, the precipitation part was at room temperature handled 3～8 minutes with the NH4CL of 140～180mmol/L again, remove red corpuscle, and then carry out centrifugal treating, and use up the pancebrin centrifuge washing again, will digest isolated cell then and dilute with physiological saline;

B, mescenchymal stem cell electrophoresis, extraction

Electrophoresis chamber evenly laterally is divided into three lattice with nylon wire screen, be 10% Repone K with weight percentage with weight percentage be 10% calglucon with 2～3: 5 volume ratio is mixed and is inserted electrophoresis chamber, the alternating-current input, voltage 90～120v, electric current 400～600mA continues 2～5 minutes, layout electrophoresis chamber in then above-mentioned gained cell solution being inserted, stir evenly, under 5～10 degrees centigrade of constant temperature, direct current input, voltage 30～50v, electric current 90～120mA, electrophoresis 15～30 minutes extracts positive electrode side electrophoresis chamber inner cell liquid, gets fat mesenchymal stem cell.

The method of this kind separation provided by the invention, purifying and extraction fat mesenchymal stem cell can overcome the above-mentioned problem of mentioning quickly and easily, and the cycle is short, and can not cause the gene that dna double revolves on the chain to sort disorderly or sudden change.

Embodiment

Below in conjunction with specific embodiment and check mensuration technical scheme of the present invention is further specified, but not as the qualification to the claimed scope of the present invention:

The method of a kind of separation provided by the invention, purifying and extraction fat mesenchymal stem cell comprises that step is as follows:

One, fat source extraction, separation, purifying

Moist liposuction method extracts the above fat of 200ML under the aseptic condition, wherein, swelling liquid preparation standard is: physiological saline 1000ML, 2% lignocaine 20ML, 0.5% suprarenin 1ML, adopt phosphoric acid salt (PBS) damping fluid to soak then, wash 3 times, digested 30 minutes in 37 degree water-bath vibrations with the serum-free DMEM nutritive medium that contains 0.1%I Collagen Type VI enzyme again, digest is the nylon mesh of 50uM more successively by the aperture, collect filtered liquid, (3000 rev/mins of centrifugal speeds) abandoned supernatant and buoyant mature fat cell in centrifugal 2 minutes, obtain matrix microtubule layer, the precipitation part was at room temperature handled 5 minutes with 160mmol/L NH4CL, remove red corpuscle, centrifugal treating uses up the pancebrin centrifuge washing 3 times then again, to digest isolated cell again and dilute with physiological saline, total amount is added to 1000ML.

Two, mescenchymal stem cell electrophoresis, extraction

Is that 50uM nylon wire screen evenly laterally is divided into three lattice with the electrophoresis chamber of 40cm * 22cm * 9cm with the aperture, with the mixed electrophoresis chamber of inserting of 10% Repone K 20ML, 10% calglucon 50ML, the alternating-current input, voltage 100v, electric current 500mA continues after three minutes, the 1000ML cell solution of above-mentioned gained is inserted middle layout electrophoresis chamber, stirred evenly 2 minutes, under 5-10 degree centigrade of constant temperature, direct current output, voltage 40v, electric current 100mA, electrophoresis 20 minutes extracts positive electrode side electrophoresis chamber inner cell liquid, gets fat mesenchymal stem cell.

Below the mescenchymal stem cell of present embodiment gained is measured:

The cell that extracts at the in-vitro separation fatty tissue mainly is divided into two kinds, and the one, sophisticated adipocyte, the fatty tissue of humans and animals is about 1st/3rd, adipocyte, volume is bigger, floats on the upper strata after centrifugal; Another kind is exactly the centrifuged deposit part, account for 2/3rds, be also referred to as the microtubule matrix components, the cell type that it comprised is more, is mainly capillary blood vessel, nerve fiber, inoblast and and be in the Preadipocyte In Vitro of various different differential periods and has stem cell of multidirectional differentiation capability etc.This stem cell also can be divided into scleroblast, chondrocyte and myocyte etc. under specific inductive condition, shown the differentiation capability of the pluripotency of this cell, and its behavior is similar to mesenchymal stem cells MSCs.

For any type stem cell, the separation of stem cell, purifying are further investigations and use the prerequisite of such stem cell that the specific molecular marker of stem cell surface is separation, purifying and identifies the important evidence of such stem cell.Present embodiment adopts negative sign and positive sign to combine, and is aided with morphology and differentiation characteristic carries out the evaluation of mescenchymal stem cell.

Assay shows that the last enchylema that extracts is spindle cell substantially in the present embodiment, and vitro culture confirms that isolated cells has stronger multiplication capacity, and the isolated cells surface marker CD44 and the CD105 positive are indicated as mescenchymal stem cell.CD14 and CD34 are that hematopoietic lineage and endothelial cell marker HLA-DR are negative, can get rid of possibility for hematopoietic cell and endotheliocyte, S-100 is that the special mark of Preadipocyte In Vitro also is negative, though point out this cell to be the fatty tissue source, but wherein Preadipocyte In Vitro is removed after centrifugal, purifying, electric field transformation, thereby the proof cell that present embodiment extracted is a mescenchymal stem cell.

Claims

1. the method for a separation, purifying and extraction fat mesenchymal stem cell is characterized in that, mainly may further comprise the steps:

A, fat source extraction, separation, purifying

Fat source extracts under the aseptic condition, soak, wash by damping fluid then, adopt the serum-free DMEM nutritive medium water-bath vibration digestion that contains 0.1%I Collagen Type VI enzyme again, get filtered liquid after the gained digest sieves and carry out centrifugal treating, obtain its matrix microtubule layer, the precipitation part is removed red corpuscle, and then uses the serum-free DMEM nutritive medium centrifuge washing that contains 0.1%I Collagen Type VI enzyme after the centrifugal treating, will digest isolated cell and dilute with physiological saline;

B, mescenchymal stem cell electrophoresis, extraction