CN104560862B - Isolated adipose tissue living cells method, medical composition and application thereof, cell bank - Google Patents
Isolated adipose tissue living cells method, medical composition and application thereof, cell bank Download PDFInfo
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Abstract
The invention discloses a kind of isolated adipose tissue living cells methods, medical composition and application thereof, cell bank, it is existing to hold cutter chopping adipose tissue as the previous step of living cells in isolated adipose tissue to solve, it is only capable of isolating a small amount of living cells and has the shortcomings that lower cell survival rate；The method comprise the steps that providing an adipose tissue；Aforementioned adipose tissue homogeneous is shredded using a cutter device；A reagent is added in the aforementioned adipose tissue shredded to be hydrolyzed；Further centrifuge separation；Supernatant is removed, to obtain a cell precipitate；Whereby, the time of chopping adipose tissue can be shortened, and cutter reuse is avoided to pollute, and improve from the adipose tissue of per unit weight and obtain viable count, and do not reduce cell survival rate, the purposes as isolated adipose tissue living cells.
Description
Technical field
The present invention relates to a kind of isolated adipose tissue living cells methods, medical composition and application thereof, cell bank, especially
Refer to that a kind of one cutter device of utilization shreds adipose tissue fast homogeneous, is further isolated from adipose tissue with benefit living thin
Born of the same parents to improve the adipose tissue acquisition viable count from per unit weight, and do not reduce cell survival rate.
Background technique
Press, the source of mesenchymal stem cell can be separated from many different tissues of human body, such as by perform the operation directly cut off or
It is stem cell source abundant via the adipose tissue that liposuction obtains, may separate out fat stem cell (Adipose
Tissue-derived stem cells, ADSCs), had the advantage that: acquisition mode is invasive low, to human injury
Amount that is small and once obtaining is more, can be in external hyperplasia culture etc.；In addition fat stem cell, which also possesses, can be divided into os osseum, soft
The potentiality (Zuk et al., 2002) of bone, muscle and fat cell, therefore, fat stem cell are considered as most research hair
Open up one of the stem cell of potentiality.
Now for perform the operation directly cut off obtained by bulk adipose tissue chopping processing mode there has been no it is a set of suitably certainly
Dynamicization system uses to arrange in pairs or groups, and generally shreds processing mode just like No. 201331366 one kind of TaiWan, China patent " in vitro without blood
Clear adult stem cell amplifies culture technique ", underwent operative is disclosed in method cut rouge and obtain adipose tissue block, and further utilize hand
Art cutter acts on after shredding the adipose tissue with Collagenase ferment, then can obtain stromal vascular confluent monolayer cells (Stromal through separation
Vascular fraction, SVF), it is the common combinations such as stroma cell, blood cell, vascular endothelial cell and fat stem cell
It forms.Only, using surgical knife tool chopping adipose tissue the step of, need to expend more time is just able to use up adipose tissue can
The chopping of energy, to obtain more stromal vascular confluent monolayer cells；And the time for operating comminution step is longer, to aforementioned stromal vascular layer
The survival rate of cell is bigger, so that acquired its survival rate of adipose stromal stem cell is relatively low.In addition, operator
Member is easier to generate the risk of specimen cross contamination because reusing surgical knife tool when operating a separate sources specimen.
For another No. 201235471 " cells of the stem cell (OFSC) comprising eye socket adipose-derived of TaiWan, China patent
The separation and application of group and they " discloses in method and collects and obtain in the orbital structures directly removed by eye socket inner cavity, or by
Entropion, ectropion of lid, ptosis or eye pouch eyelid moulding operation it is collected obtained by eye socket fat, can easily use
Scissors or tweezers dismember the eye socket adipose tissue and act on again with Collagenase ferment, then are filtered, are centrifugated to obtain
Cell precipitate, and the cell precipitate is subjected to cell culture to obtain the cell with group's Forming ability.Only, the case institute
The method of announcement, which has been equally existed, dismembers the eye socket adipose tissue using scissors or tweezers, need to expend more time be just able to by
Adipose tissue shreds as far as possible, to obtain more cell precipitate；And relatively, the time for operating comminution step gets over
Long, the influence to the survival rate of cell in aforementioned cells sediment is bigger.
In summary, above-mentioned existing using scarce brought by surgical knife tool progress adipose tissue chopping operation in order to solve
Lose, there is an urgent need for the methods that one can quickly shred adipose tissue to separate living cells, under the premise of not reducing cell survival rate, with into
One step improves from the adipose tissue of per unit weight and obtains number of viable cells.
Summary of the invention
The purpose of the present invention is to provide a kind of isolated adipose tissue living cells method, medical composition and application thereof, carefully
Born of the same parents library, to achieve the purpose that the adipose tissue acquirement viable count improved from per unit weight and do not reduce cell survival rate.
In order to achieve the above objectives, solution of the invention is:
A kind of isolated adipose tissue living cells method comprising have the following steps:
Step a provides an adipose tissue；
The adipose tissue is placed in the closed container of a cutter device by step b；
Step c makes the cutter device bestow an active force to the closed container, enables one in the closed container default cutting
Cutter executes fast homogeneous chopping to the adipose tissue, obtains one and homogenizes adipose tissue；
Step d homogenizes in this aforementioned shredded and a reagent is added in adipose tissue, and reaction is hydrolyzed；
Step e is filtered aforementioned, be centrifugated through processed this of the hydrolysis adipose tissue that homogenizes；And
Step f removes the supernatant after centrifuge separation, to obtain a cell precipitate.
It may include aforementioned adipose tissue is cleaned with phosphate buffer solution the step of before above-mentioned steps a.
Adipose tissue described in above-mentioned steps a is the bulk fat got from mammal surgical resection
Tissue.
Above-mentioned mammal is the mankind.
Cutter device described in above-mentioned steps b is a deserted homogenizer.
Above-mentioned deserted homogenizer includes a deserted closed container and a power unit, and the deserted closing is held
There is a cutting tool in device.
Above-mentioned steps c homogeneous chopping condition is built into 1 g to 6 g for the deserted closed container in 15 milliliters of volumes
Adipose tissue carries out fast homogeneous chopping.
Above-mentioned steps c homogeneous shreds preferable condition and is built into 3 g of rouge for the deserted closed container in 15 milliliters of volumes
Fat tissue carries out fast homogeneous chopping.
The revolving speed of above-mentioned power unit is 300 revolutions per minutes to 3000 revolutions per minutes, and action time is 3 minutes
To 10 minutes.
The preferable revolving speed of above-mentioned power unit is 600 revolutions per minutes to 1500 revolutions per minutes.
Reagent described in above-mentioned steps d be selected from a trypsase, a neutral proteinase, a glue enzyme, a sodium hyaluronate enzyme,
One or a combination set of one first collagen type enzyme or one the 4th the constituted group of collagen type enzyme.
Hydrolysis described in above-mentioned steps d is that whole process is placed in a constant temperature hybridization reaction baking oven and carries out, and condition is
Four degrees celsius to 45 degree, revolving speed is 5 revolutions per minutes to 50 revolutions per minutes and the time 0.5 hour to 24 hours.
Above-mentioned preferable condition be it is 37 degree Celsius, revolving speed is 15 revolutions per minutes and the time 8 hours.
The condition of centrifugation described in above-mentioned steps e is 400 × g, the time 10 minutes.
Above-mentioned steps e is further included will filter out adipose tissue disintegrating slag after gained one filtered fluid move into a centrifuge tube the step of,
To be centrifuged.
Cell precipitate described in above-mentioned steps f is a stromal vascular confluent monolayer cells.
A step g is further included after above-mentioned steps f phosphate buffer solution is added in aforementioned cells sediment, clean aforementioned
Cell precipitate, the cell precipitate being centrifuged after obtaining a cleaning to remove the supernatant containing watery blood and aforementioned agents again.
A step h is further included after above-mentioned steps g to be cultivated in cell precipitate one culture medium of merging after aforementioned cleaning,
To obtain the cell of an amplification.
Above-mentioned culture medium includes: a basal medium, a serum additive, a second type fiber mother cell growth factor.
Above-mentioned serum additive is a fetal calf serum, and the concentration expressed in percentage by volume used is percent 2 to percent 10, and
The second type fiber mother cell growth factor is 1 ng/ml to 20 ngs/ml using concentration.
The cell of above-mentioned amplification is the undifferentiated adipose stromal stem cell of essence.
Above-mentioned adipose stromal stem cell is at least performance cell surface antigen CD90 or CD105 or combinations thereof, and not table
The cell mass of existing CD45.
Above-mentioned adipose stromal stem cell is one of material, regenerative medicine or the cellular system engineering applied to cell therapy
Or combinations thereof.
It is a kind of dry comprising the aforementioned undifferentiated adipose stromal of essence acquired in above-mentioned isolated adipose tissue living cells method
Cell is applied to degenerative disorders, repair tissue damage, neomorph in the purposes for the medical composition for preparing cell therapy
Or one or a combination set of regeneration.
It later include the adipose stromal stem cell of an aforementioned amplification of step i freezen protective in above-mentioned steps h, for more into one
The application of step.
A kind of cell bank, it includes the adipose stromal stem cells of the amplification by above-mentioned steps i institute freezen protective.
It include that a step i` executes the adipose stromal stem cell that aforementioned amplification is broken up in induction after above-mentioned steps h, to obtain
Take a cell broken up from the adipose stromal stem cell of aforementioned amplification.
The cell broken up from the adipose stromal stem cell of above-mentioned amplification, including osteocyte, fat cell or cartilage cell
One of.
A kind of medical composition, it includes dry from aforementioned adipose stromal acquired in above-mentioned isolated adipose tissue living cells method
One or a combination set of undifferentiated adipose stromal stem cell of cell or aforementioned essence that cell is broken up.
After adopting the above structure, effect of the invention is:
Shorten chopping adipose tissue step the time required to: the present invention by using preceding cutting device can it is more existing with operation
The method that cutter executes chopping more shortens chopping adipose tissue step required time, and can reduce between operator because skill is ripe
Practice influence caused by difference, to lower cell in adipose tissue because the cell death caused by leaving human body overlong time shows
As, and then improve cell survival rate.
Improve living cells yield rate: the present invention is shredded adipose tissue homogeneous by using preceding cutting device, to increase
The contact area of adipose tissue and added hydrolysis reagent increases, and improves hydrolysis effect, to improve from per unit
The adipose tissue of weight obtains the number of stromal vascular confluent monolayer cells, and improves aforementioned stromal vascular confluent monolayer cells in further culture
Afterwards, resulting adipose stromal stem cell population, and do not reduce cell survival rate.
Reduce specimen cross contamination: the present invention is using a deserted closed container and a power unit to acquired
A blocky adipose tissue specimen carries out fast homogeneous comminution step, and a specimen for separate sources is respectively placed in a deserted closed container,
Operator be can avoid when operating a separate sources specimen because of the cross-contamination phenomena caused by accidentally touching.
Obtain the adipose stromal stem cell with differentiation potential: by adipose stromal stem cell acquired in the method for the present invention
It is at least to show cell surface antigen CD90 and CD105, and does not show the cell mass of CD45, and has and be divided into os osseum, soft
The ability of bone and fat keeps the pluripotency through adipose stromal stem cell acquired in the method for the present invention with differentiation potential dry thin
The abundant source of born of the same parents.
Detailed description of the invention
Fig. 1 is flow chart of the method for the present invention；
Fig. 2A is that the different adipose tissue g numbers of one contributor in the present invention can obtain stromal vascular confluent monolayer cells number ratio
Compared with figure；
Fig. 2 B to illustrate the invention in one contributor different adipose tissues g number gained stromal vascular confluent monolayer cells
Survival rate compares figure；
Fig. 2 C is in the present invention secondly the different adipose tissue g numbers of contributor can obtain stromal vascular confluent monolayer cells number ratio
Compared with figure；
Fig. 2 D to illustrate the invention in secondly contributor different adipose tissues g number gained stromal vascular confluent monolayer cells
Survival rate compares figure；
Fig. 3 A is that surgically scissors processing can with processing adipose tissue under the conditions of homogenizer different rotating speeds in the present invention
It obtains stromal vascular confluent monolayer cells number and compares figure；
Fig. 3 B is surgically scissors handles and handles adipose tissue institute under the conditions of homogenizer different rotating speeds in the present invention
The survival rate for obtaining stromal vascular confluent monolayer cells compares figure；
Fig. 4 A obtains matrix blood for the adipose tissue of 9 contributors in the present invention with the opposite surgical scissors of homogenizer chopping
The total cell yield ratio of tube layer cell compares figure；
Fig. 4 B obtains matrix blood for the adipose tissue of 9 contributors in the present invention with the opposite surgical scissors of homogenizer chopping
The survival rate ratio of tube layer cell compares figure；
Fig. 5 A, which is the adipose tissue of 9 contributors in the present invention, shreds the obtaining between fat of opposite surgical scissors with homogenizer
The total cell yield ratio of matter stem cell compares figure；
Fig. 5 B, which is the adipose tissue of 9 contributors in the present invention, shreds the obtaining between fat of opposite surgical scissors with homogenizer
The survival rate ratio of matter stem cell compares figure；
Fig. 6 A is that contributor's body-mass index and the stromal vascular confluent monolayer cells of every g of adipose tissue are got in the present invention
Rate (cell/g) relational graph；
Fig. 6 B is that contributor's body-mass index and the adipose stromal stem cell of every g of adipose tissue are got in the present invention
Rate (cell/g) relational graph；
After Fig. 7 A is induced to differentiate into os osseum cell for adipose stromal stem cell in the present invention, through alkaline phosphatase staining knot
Fruit figure；
After Fig. 7 B is induced to differentiate into os osseum cell for adipose stromal stem cell in the present invention, through Feng Kusa coloration result
Figure；
Fig. 7 C is that adipose stromal stem cell is after being induced to differentiate into cartilage cell in the present invention, through A Erxin indigo plant coloration result
Figure；
Fig. 7 D is that adipose stromal stem cell is after being induced to differentiate into fat cell in the present invention, through oil red O stain result figure；
Fig. 8 is the operation chart of step b and c in the present invention.
Symbol description
A provides an adipose tissue
The adipose tissue is placed in the closed container of a cutter device by b
C makes the cutter device bestow an active force to the closed container, enables the default cutting tool in the closed container
Fast homogeneous chopping is executed to the adipose tissue, one is obtained and homogenizes adipose tissue
D homogenizes in this aforementioned shredded and a reagent is added in adipose tissue, and reaction is hydrolyzed
E is filtered aforementioned, be centrifugated through processed this of the hydrolysis adipose tissue that homogenizes
F removes the supernatant after centrifuge separation, to obtain a cell precipitate
Phosphate buffer solution is added in aforementioned cells sediment g, cleans aforementioned cells sediment, is centrifuged again to remove
Supernatant containing watery blood and aforementioned agents, the cell precipitate after obtaining a cleaning
Cell precipitate after aforementioned cleaning is placed in a culture medium and cultivates by h, to obtain the cell of an amplification
The cell of the aforementioned amplification of i freezen protective
The cell of aforementioned amplification is broken up in i` induction, to obtain a cell broken up from the cell of aforementioned amplification
1 adipose tissue 1A homogenizes adipose tissue
The deserted closed container of 2 homogenizer 21
211 cutting tool, 22 power unit.
Specific embodiment
In order to further explain the technical solution of the present invention, being explained in detail below by specific embodiment the present invention
It states.
Firstly, please join Fig. 1, the flow chart of the method for the present invention, the present invention provides a kind of isolated adipose tissue living cells method,
It includes the following steps: that step a provides an adipose tissue, the blocky rouge got from mammal surgical resection
Fat tissue, and the present invention is using the blocky adipose tissue got from mankind's surgical resection as embodiment；Step b is by the rouge
Fat tissue 1 is placed in the step in the deserted closed container 21 of a deserted homogenizer 2, the homogenizer 2 (DT-20 gamma,
IKA ULTRA TURRAX Tube drive) include one 15 milliliters of volumes deserted closed container 21 and a power unit
22, and there is a cutting tool 211 (ginseng Fig. 8) in the deserted closed container 21；Step c abandons the power unit 22 to this
Formula closed container 21 bestows an active force, and the cutting tool 211 in the deserted closed container 21 is enabled to execute the adipose tissue 1
Fast homogeneous chopping obtains one and homogenizes adipose tissue 1A (ginseng Fig. 8), and 1 g to 6 g adipose tissue 1 is placed in by the present invention
In the deserted closed container 21, homogeneous chopping is carried out, and preferred embodiment is that this is deserted by 3 g of mergings of adipose tissues 1
In closed container 21, homogeneous chopping is carried out.It is preferred that the revolving speed of previous power unit 22 is 300 revolutions per minutes to 3000
Revolutions per minute, and action time is 3 minutes to 10 minutes；And preferably revolving speed is that 600 revolutions per minutes turn per minute to 1500
Number, optimum speed are 1200 revolutions per minutes；Step d homogenizes in this aforementioned shredded and an examination is added in adipose tissue 1A
Reaction is hydrolyzed in agent, and wherein the reagent is selected from a trypsase (Trypsin), a neutral proteinase (Dispase), a glue
Enzyme (Gelatase), a sodium hyaluronate enzyme (Hyaluronidase), one first collagen type enzyme (Collagenase type I)
Or one the 4th one or a combination set of collagen type enzyme (Collagenase type IV) constituted group；It is preferred that this hair
It is bright with the first collagen type enzyme or the 4th collagen type enzyme (Worthington Biochemical
Corporation) it is embodiment, and the use of concentration is 0.2 mg/ml to 20 mg/mls；It is preferred that aforementioned hydrolysis
Reaction whole process, which is placed in a constant temperature hybridization reaction baking oven (MO-01, Double Eagle Enterprise), to be carried out, and condition is
4 degree to 45 degree of temperature in degree Celsius, revolving speed are 5 revolutions per minutes to 50 revolutions per minutes and the time 0.5 hour to 24 hours, and most
Good condition is 37 degree of temperature in degree Celsius, revolving speed is 15 revolutions per minutes and the time 8 hours.Step e by it is aforementioned through hydrolysis at
The adipose tissue managed filters out adipose tissue disintegrating slag first to obtain a filtered fluid, and wherein foregoing filtration mode can be with any technology
Known mode (such as filter membrane or strainer) carries out to obtain foregoing filtration liquid in field；The filtered fluid is moved into one 50 milliliters again
The centrifuge tube of volume is centrifuged, and the condition of aforementioned centrifugation be 400 × g, the time 10 minutes；And after step f will be separated
Supernatant remove, to obtain a cell precipitate, which is a stromal vascular confluent monolayer cells (Stromal
Vascular Fraction, SVF), by a stroma cell, a blood cell, a vascular endothelial cell and an adipose stromal
Stem cell is formed.
Aforementioned cells sediment is added in phosphate buffer solution it is preferred that further including a step g after abovementioned steps f
In, aforementioned cells sediment is cleaned, is centrifuged again to remove the supernatant containing watery blood and aforementioned agents, after obtaining a cleaning
Cell precipitate.It is preferred that further including a step h after abovementioned steps g for the cell precipitate merging one after aforementioned cleaning
It is cultivated in culture medium, to obtain the undifferentiated adipose stromal stem cell of essence of amplification.It is preferred that aforementioned adipose stromal is dry
Cell be cell surface antigen CD45-, CD90+and CD105+cell mass.It is preferred that prior culture media packet
Contain: a basal medium (IMDM), a serum additive, a second type fiber mother cell growth factor；Wherein, which adds
Object is a fetal calf serum, and the concentration expressed in percentage by volume used is percent 2 to percent 10, and the second type fibroblast is raw
The long factor is 1 ng/ml to 20 ngs/ml, preferably 10 ngs/ml using concentration.
It is preferred that include the adipose stromal stem cell of an aforementioned amplification of step i freezen protective after the abovementioned steps h,
For further applying, such as the research of clinic study, regenerative medicine or the development of cellular system engineering.Preferably
Being also can include that a step I` executes the adipose stromal stem cell that aforementioned amplification is broken up in induction after abovementioned steps h, to obtain
The cell broken up from the adipose stromal stem cell of aforementioned amplification, and the cell of aforementioned differentiation includes osteocyte, fat cell
Or one of cartilage cell.
Present invention simultaneously provides a kind of cell bank, the adipose stromal of the amplification comprising abovementioned steps i institute freezen protective is dry thin
Born of the same parents.
It is preferred that " freezen protective " word as used herein typically refers to cell cryoprotector such as diformazan is added
The method that subzero temperature saves, such as be negative 80 DEG C or minus 196 DEG C of (liquid nitrogens are cooled to after sulfoxide (DMSO) or glycerol
Boiling point).Freezen protective can be such as method and process that those skilled in the art is carried out, and only non-present invention demand emphasis is therefore
It will not go into details (refering to HummaPress company in 1997 published second edition Pollard, J.W. and Walker, base written by J.M..
Plinth cell culture process；Wiley-Liss company in 2000 publishes the 4th edition Freshney, R.I., written by zooblast training
It supports).
The present invention provides a kind of medical composition again, and the adipose stromal for being obtained from aforementioned amplification it includes abovementioned steps i` is dry
One or a combination set of undifferentiated adipose stromal stem cell of cell or aforementioned essence that cell is broken up.
It is preferred that aforementioned medical composition includes aforementioned adipose stromal stem cell, from aforementioned adipose stromal stem cell institute
The one of a cell secreta secreted by the cell of differentiation, aforementioned adipose stromal stem cell or aforementioned adipose stromal stem cell is thin
One or a combination set of born of the same parents' extract treats a upper acceptable carrier/excipient with suitable.It is preferred that known the technology
The those skilled in the art person in field all knows, any in principle to point out the suitable shape treated by Human Meta stem cell via research report
Condition or disease can all be treated by medical composition of the invention.It wherein seem that stem cell transplantation can be effectively as specific
Therapeutical uses；For example, the stem cell for generating hematopoiesis race can be used for replacing the hemopoietic system in marrow；Generate the dry thin of interstitial family
Born of the same parents can be used for repairing musculoskeletal disease；Stem cell, which is divided into epithelium family person, can be used for repairing surface damage；Stem cell differentiation
For nerve cell, family person can be used for treating neurodegenerative disorders.
It is preferred that adipose stromal stem cell acquired in method of the invention is at least to show cell surface antigen
CD90 and CD105, and the cell mass of CD45 is not showed, and there is the potential for being divided into os osseum, cartilage and fat simultaneously, make through this
Adipose stromal stem cell acquired in inventive method is the abundant source of the pluripotent stem cell with differentiation potential, and has simultaneously
There is the potential clinical application of cell therapy.Therefore, adipose stromal stem cell acquired in method of the invention can be applied to carefully
On material, regenerative medicine and the cellular system engineering that born of the same parents treat, such as future can be used in treatment or delay degenerative disorders, repair
Overlying tissue damage, neomorph or regeneration.
The embodiment of the present invention is that the embodiment of the blocky adipose tissue got with being derived from mankind's surgical resection is given
It is illustrated with demonstration, but the present invention is not limited by following embodiments.
" experiment one " isolated adipose tissue mesostroma blood vessel confluent monolayer cells
1. cell number and cell survival rate that every g of adipose tissue can obtain stromal vascular confluent monolayer cells
This experiment belonging to the Buddhism Tzu Chi general hospital by the gene authenticated of ISO14644 Class 7 and stem cell again
It is carried out in raw laboratory, and the training of this experiment operator is that human cell tissue superior operational specification (GTP) is followed to carry out.
The present invention collects the abdominal subcutaneous adipose tissues block specimen from 11 caesarean birth contributors of Min Sheng hospital, and weight is respectively about 30 public
Gram, and the specimen that the present invention is collected is to ratify to carry out by Min Sheng hospital internal examination board.First donated with wherein two
The specimen of person carries out this experiment, and it is public that the specimen weight of one contributor is divided into 1 g, 2 g, 3 g, 4 g, 5 g and 6
Grams equal six groups, and secondly the specimen weight of contributor is divided into 1 g, 2 g, 3 g, 4 g, 5 g etc. five groups, divided
From, as separating step is as previously described, repeat no more, and cell survival rate measurement part is by resulting stromal vascular layer out of the ordinary
Cell with the third pyridine of monoiod(in)ate (Propidium Iodide, PI) dye after, through an automated cell calculating instrument (Adam-MC,
NanoEnTek) come calculate each group it is separated go out stromal vascular confluent monolayer cells number；The cell number of another stromal vascular confluent monolayer cells measures
First cell membrane is broken with a cellular membrane lysis buffer solution, then with the DNA of propidium iodide dye nucleus, whereby to avoid the base
The red blood cell that matter blood vessel confluent monolayer cells are included influences the counting of the stromal vascular confluent monolayer cells number, more accurate thin to obtain
Born of the same parents' number measured value.
1.1 experimental result
This experimental data is the level of signifiance with p < 0.05 through Microsoft Excel Software t test (t-test) statistical analysis,
And result is quantified as statistical chart.Please referring initially to Fig. 2A, the different adipose tissue g numbers of aforementioned one contributor can obtain matrix
Vascular lamina cell number compares figure, by as a result, it has been found that, take 3 g of adipose tissues to be placed in the deserted closing of aforementioned 15 milliliters of volumes
That group of homogeneous chopping is carried out in container, the cell number that every g of adipose tissue can obtain stromal vascular confluent monolayer cells is more than 2.5
× 105, be highest one group；Continuous ginseng Fig. 2 B, the different adipose tissues g number gained stromal vascular of aforementioned one contributor
Confluent monolayer cells survival rate compares figure, can be found by result, and 3 g of adipose tissues is taken to be placed in the deserted closing of aforementioned 15 milliliters of volumes
That group of homogeneous chopping is carried out in container, it is higher one group that cell survival rate, which is more than percent 80,.Another ginseng Fig. 2 C, it is aforementioned
Secondly the different adipose tissue g numbers of contributor, which can obtain stromal vascular confluent monolayer cells number, compares figure, can be found by result, equally
3 g of adipose tissues are taken to be placed in that group for carrying out homogeneous chopping in the deserted closed container of aforementioned 15 milliliters of volumes, every public affairs
It is more than 7.0 × 104 that gram adipose tissue, which can obtain the cell number of stromal vascular confluent monolayer cells, is highest one group；Continuous ginseng Fig. 2 D, it is preceding
State secondly contributor different adipose tissues g number gained stromal vascular confluent monolayer cells survival rates compare figure, can be found by result,
3 g of adipose tissues are taken to be placed in that group for carrying out homogeneous chopping in the deserted closed container of aforementioned 15 milliliters of volumes, cell
Survival rate about percent 90 is higher one group.It is learnt by above-mentioned experiment, takes aforementioned 15 milli of 3 g of adipose tissue block mergings
Rising and carrying out homogeneous chopping in the deserted closed container of volume is preferable condition, can obtain the matrix blood in every g of adipose tissue
The preferable cell number and preferable cell survival rate of tube layer cell.
2. surgical cutter is deposited with homogenizer different rotating speeds conditions on cell number and cell deserted in the present invention
The influence of motility rate is compared
This experiment is the adipose tissue block for taking aforementioned one contributor, and be divided into surgical operation scissors group, 400 groups of revolving speed,
Five groups of 600 groups of revolving speed, 1200 groups of revolving speed and 1500 groups of revolving speed etc., carry out the homogeneous comminution step of adipose tissue, with benefit is subsequent should
The separation of stromal vascular confluent monolayer cells, and subsequent separating step is identical as preceding method；And the cell survival rate measurement side of this experiment
Method and the cell number measuring method of stromal vascular confluent monolayer cells are all identical as previous experiments, repeat no more.
2.1 experimental result
This experimental data is the level of signifiance with p < 0.05 through Microsoft Excel Software t test (t-test) statistical analysis,
And result is quantified as statistical chart.Join shown in Fig. 3 A, surgical operation scissors is handled and handled under the conditions of the homogenizer different rotating speeds
Adipose tissue can obtain stromal vascular confluent monolayer cells number and compare figure.As can be seen from the results, it in that group of the homogenizer revolving speed 1200, obtains about
1.2 × 106 cell numbers are highest one group, can be obtained cell number far more than the surgical operation scissors group.Continuous ginseng
Fig. 3 B, the processing of surgical operation scissors and stromal vascular confluent monolayer cells obtained by processing adipose tissue under the conditions of the homogenizer different rotating speeds
Survival rate compares figure.As can be seen from the results, in that group of the homogenizer revolving speed 1200, the survival rate of obtained stromal vascular confluent monolayer cells
About percent 80, it is higher one group, and the survival rate of the obtained stromal vascular confluent monolayer cells of surgical operation scissors group is not
To percent 70.By above-mentioned experimental result it is found that by the method for the present invention with the homogenizer replace existing surgical knife tool into
The homogeneous of row adipose tissue block shreds operation, the cell number of obtained stromal vascular confluent monolayer cells not only can be improved, and again not
The cell survival rate of the stromal vascular confluent monolayer cells can be reduced.
3. deserted homogenizer is handled under opposite surgical scissors processing, the total cell of gained stromal vascular confluent monolayer cells is obtained
Rate ratio is compared with survival rate ratio
This experiment is the subcutaneous abdomen bulk adipose tissue specimen using aforementioned 9 contributors, is respectively divided into surgical scissors group
With 1200 groups of homogenizer revolving speed etc. two groups, each group takes 3 g of adipose tissue blocks to carry out homogeneous chopping operations, with the subsequent matrix of benefit
The separation of blood vessel confluent monolayer cells, and subsequent separating step is identical as preceding the method.The cell survival rate measuring method of this experiment with
The cell number measuring method of stromal vascular confluent monolayer cells is all identical as previous experiments, repeats no more；Only, acquired results are converted to
Matter machine/surgical scissors ratio (again) mode is presented.
3.1 experimental result
This experimental data is the level of signifiance with p < 0.05 through Microsoft Excel Software t test (t-test) statistical analysis,
And result is quantified as statistical chart.Join Fig. 4 A, the adipose tissue of 9 contributors shreds opposite operation with the homogenizer in the present invention
The total cell yield ratio (again) for obtaining stromal vascular confluent monolayer cells of scissors compares figure.It can be found by result, be donated at aforementioned 9
The adipose tissue of person in the process of the present invention in the homogenizer shred opposite surgical scissors obtain the total thin of stromal vascular confluent monolayer cells
For born of the same parents' yield ratio (again) between 1.3 times to 10.2 times, ratio size is different because of individual.It is continuous to join Fig. 4 B, 9 donations in the present invention
The adipose tissue of person shreds the cell survival rate ratio of the obtained stromal vascular confluent monolayer cells of opposite surgical scissors with the homogenizer
(again) compares figure.Can be found by result, aforementioned 9 contributors adipose tissue in the process of the present invention in the homogenizer shred phase
To the cell survival rate ratio (again) of the obtained stromal vascular confluent monolayer cells of surgical scissors between 0.9 times to 2.3 times, ratio size
It is different because of individual.It follows that replacing existing surgical knife tool to carry out adipose tissue block with the homogenizer in method of the invention
Homogeneous shreds operation, the cell number of obtained stromal vascular confluent monolayer cells not only can be improved, and will not reduce the matrix blood again
The cell survival rate of tube layer cell.
4. deserted homogenizer is handled under opposite surgical scissors processing, the total cell of gained adipose stromal stem cell is obtained
Rate ratio is compared with survival rate ratio
The packet mode of this experiment is identical as the 3. of aforementioned " experiment one ", and further by the resulting stromal vascular layer of each group
Cell culture is 5 days in a culture medium, to obtain adipose stromal stem cell, and analyzes homogenizer and handles opposite surgical scissors processing
Under, the influence to the total cell yield and survival rate of gained adipose stromal stem cell.Prior culture media includes: a fetal calf serum
The basis of additive (GIBCO-Invitrogen), a second type fiber mother cell growth factor (FGF-2, R&D Systems)
In culture medium (IMDM, GIBCO-Invitrogen)；Wherein, the concentration expressed in percentage by volume which uses is hundred
/ 2 to percent 10, which is 10 ngs/ml using concentration.Every group of matrix
Blood vessel confluent monolayer cells are incubated in cell culture with the cell density of every square centimeter of 10000 cells (cells/cm2)
(Becton Dickinson), and all cells are all incubated at 37 degree Celsius, percent 5 carbon dioxide partial pressures and percent 95 wet
Spend the incubator (Forma Series II Model 3110, Thermo) under environment, and every 3 days one subcultures of replacement.It will
After each group adipose stromal stem cell was cleaned once after culture 5 days with phosphate buffer solution (PBS), with trypsase-second two
Amine tetraacethyl (Trypsin-EDTA, GIBCO-Invitrogen) solution is in 37 degree Celsius effects after five minutes again with cell spatula
It carefully removes and is not applied complete cell, be added in culture medium of the equal proportion containing fetal calf serum and the ferment of trypsase acts on
Activity.The measurement of cell survival rate is with a cell counter (Vi-CELL AS, Beckman Coulter) calculating.Survival
Cell is distinguished with 0.4% trypan blue (Trypan-blue, GIBCO-Invitrogen) with dead cell, and when calculating determines to live
Interstital stem cell parameter setting are as follows: 100 images, 10 to 30 microns of size, 75% live brightness, 5% field regions.
The adipose stromal stem cell of another each group, which gets rate, to be calculated using the formula of doubling time (DT), DT=t/ (3.32
[log10Nt-log10N0]), wherein Nt refers to cell final density, and N0 refers to cell initial density.This experiment is equally by gained
As a result homogenizer/surgical scissors ratio (again) mode is converted to present.
4.1 experimental result
This experimental data is the level of signifiance with p < 0.05 through Microsoft Excel Software t test (t-test) statistical analysis,
And result is quantified as statistical chart.Join Fig. 5 A, the adipose tissue of 9 contributors shreds opposite operation with the homogenizer in the present invention
The total cell yield ratio for obtaining adipose stromal stem cell of scissors compares figure.By as the result is shown it is found that in aforementioned 9 contributors
Adipose tissue in the process of the present invention in the homogenizer shred the total cells for obtaining adipose stromal stem cell of opposite surgical scissors
For yield ratio (again) between 1.2 times to 7.8 times, ratio size is different because of individual.It is continuous to join Fig. 5 B, 9 contributors in the present invention
Adipose tissue shredded with the homogenizer opposite surgical scissors obtained adipose stromal stem cell cell survival rate ratio (again)
Compare figure.Can be found by result, aforementioned 9 contributors adipose tissue in the process of the present invention in the homogenizer shred opposite hand
The cell survival rate ratio (again) of the obtained adipose stromal stem cell of art scissors is between 0.8 times to 4.8 times, and ratio size is because a
Body and it is different.It follows that replacing the homogeneous of existing surgical knife tool progress adipose tissue block in method of the invention with the homogenizer
Operation is shredded, the cell number of obtained adipose stromal stem cell not only can be improved, and it is dry to reduce the adipose stromal again
The cell survival rate of cell.
5. between the height and weight index (BMI) of contributor and every g of adipose tissue mesostroma blood vessel confluent monolayer cells and fat
Matter stem cell gets rate relationship
This experiment is for the height and weight index of aforementioned 9 contributors and aforementioned deserted homogenizer processing and existing outer
Section's surgical scissors handle experimental results and combine, further to analyze height and weight index and every g of adipose tissue mesostroma
Blood vessel confluent monolayer cells and adipose stromal stem cell get rate relationship.
5.1 experimental result
Join Fig. 6 A, the stromal vascular confluent monolayer cells of 9 contributor's height and weight indexes and every g of adipose tissue in the present invention
Get rate (cell/g) relational graph.The each point of homogeneous unit deserted in the present invention in figure is through one R2 of calculated
0.0794 trend dotted line, and each point of the surgical operation scissors group, the trend solid line for being 0.0155 through one R2 of calculated,
It, can be with it will thus be seen that method of the invention compares surgical operation scissors group when contributor's height and weight index is relatively low
More stromal vascular confluent monolayer cells are obtained from every g of adipose tissue.It is continuous to join Fig. 6 B, 9 contributor's height and weights in the present invention
The adipose stromal stem cell of index and every g of adipose tissue gets rate (cell/g) relational graph.Throwing in the present invention in figure
The each point of the homogeneous unit of abandoning formula, the trend dotted line for being 0.3262 through one R2 of calculated, and the surgical operation scissors group
Each point, the trend solid line for being 0.2146 through one R2 of calculated, it will thus be seen that method of the invention compares scissors for surgery
It is dry can to obtain more adipose stromal when contributor's height and weight index is relatively high from every g of adipose tissue for knife group
Cell.
" experiment two " analysis and application through the method for the present invention adipose stromal stem cell obtained
1. the Cell surface antigen analysis of typical mesenchymal stem cell
This experiment is to carry out cell surface antigen with flow cytometer (FACSCalibur, Becton Dickinson)
Measurement.It will be after the method for the present invention adipose stromal stem cell squamous subculture obtained, with trypsin-EDTA
(Trypsin-EDTA) solution is removed in 37 degree Celsius effects attachment removal in 5 minutes, and to be centrifuged after phosphate buffer clean and reuse
Supernatant is to obtain a cell precipitate, then back dissolving is in suitable phosphate buffer, respectively to different antigen with corresponding
Immunofluorescence Primary antibodies dyed, including CD13, CD34, CD44, CD45, CD73, CD90, CD105, β2Microglobulin
(B2M) and the antibody (Becton Dickinson) such as HLA-DR.It is protected from light after dyeing 15 minutes at room temperature, appropriate phosphorus is added
Upper machine analysis after acid buffer, after flow cytometer collects data, with flow cytometry analysis software (FACSCalibur,
Becton Dickinson) it is analyzed.Wherein negative control group is to omit the staining procedure of above-mentioned Primary antibodies.
1.1 experimental result
Join following table one, by result it can be found that through the method for the present invention adipose stromal stem cell obtained, cell race
Group be CD13+, CD34-, CD44+, CD45-, CD73+, CD90+, CD105+, for similar interstital stem cell
Cell populations, that is, indicate to say, still possess through the method for the present invention adipose stromal stem cell obtained similar to interstital stem cell
Surface antigen feature.
One, Cell surface antigen analysis of table
| Cell surface antigen | Percentage (%) |
| CD13 | 100.0 |
| CD34 | 2.0 |
| CD44 | 99.9 |
| CD45 | 0.1 |
| CD73 | 99.9 |
| CD90 | 100.0 |
| CD105 | 97.4 |
| HLA-DR | 0.3 |
2. the differentiation of adipose stromal stem cell
Reported in literature points out that it seems fat and bone that adipose stromal stem cell, which has, which is divided into mesoblastemic ability,
Cell.This experiment is after adipose stromal stem cell is suitably induced differentiation culture, it to be made to be divided into os osseum cell, cartilage cell
And fat cell, to confirm through the method for the present invention adipose stromal stem cell obtained, if still have stem cell multifunctional
Differentiation capability.Induction Analytical Chemical Experiment in the present invention is according to the stem cell induction differentiation system being commonly used in existing literature
System (Kanda et al., 2011；Song et al., 2010), main because therefore it will not go into details for non-this case demand emphasis
Purpose is in confirmation through the method for the present invention adipose stromal stem cell obtained, if still has stem cell multifunctional differentiation potency
Power.The os osseum cell of differentiation is dyed, the alkaline phosphatase with alkaline phosphatase (Alkaline phosphatase, ALP)
For mature osteoblast differentiation important indicator, and its colouring method be according to existing staining technique carry out (Yoshimura et
Al., 2011), details are not described herein；Existing Feng Kusa (Von-kossa) dyeing is separately carried out again, has calcium phosphate with confirmation
Presence.The cartilage cell of differentiation is to confirm the egg possessed in cartilaginous tissue with A Erxin indigo plant decoration method (Alcian blue)
The presence (Song et al., 2010) of white polysaccharide (Proteoglycan).The fat cell of differentiation is with oil red O stain (Oil
Red O staining), be confirmed whether to have lipid vacuole (Lipid vacuoles) presence (Kanda et al.,
2011)。
2.1 experimental result
Join Fig. 7 A, is alkaline phosphatase staining as a result, scale bar is 500 microns (μm), it is obtained through the method for the present invention
Adipose stromal stem cell is induced to differentiate into os osseum cell, after alkaline phosphatase staining, there is the part that black crystalline is presented,
That is, represent the presence of alkaline phosphatase；Continuous ginseng Fig. 7 B, is Feng Kusa (Von-kossa) coloration result, and scale bar is 500 micro-
Rice, it is possible to find the calcium phosphate crystal for presenting black or dark brown is again illustrated through between the method for the present invention fat obtained
Matter stem cell has the ability for being divided into os osseum cell.Join Fig. 7 C again, be A Erxin indigo plant coloration result, scale bar is 500 micro-
Rice, it is possible to find show the proteoglycan coloration result of blue, represent through the method for the present invention adipose stromal stem cell obtained
With the ability for being divided into cartilage cell.Continuous ginseng Fig. 7 D is oil red O stain as a result, scale bar is 500 microns, it is possible to find has and is in
Reveal red lipid vacuole coloration result, that is, represents to have through the method for the present invention adipose stromal stem cell obtained and break up
For the ability of fat cell.Via above-mentioned differentiated result it can be seen that, it is true through the method for the present invention adipose stromal stem cell obtained
It is real that there is stem cell multifunctional differentiation capability.
In summary experimental result is it is found that replace existing surgical knife tool in method of the invention with a deserted homogenizer
The homogeneous for carrying out adipose tissue block shreds operation, the cell number of obtained adipose stromal stem cell not only can be improved, and again
The cell survival rate of the adipose stromal stem cell will not be reduced.It is separately certain through the method for the present invention adipose stromal stem cell obtained
Cell surface antigen feature and multi-functional differentiation capability with interstital stem cell, enable the method for the present invention fat obtained
Interstital stem cell is the abundant source of the pluripotent stem cell with differentiation potential, and has cell therapy potentially clinical simultaneously
Application possibility.Further aforementioned adipose stromal stem cell can be used as medical component is prepared whereby, it is thin to be applied to
On material, regenerative medicine and the cellular system engineering that born of the same parents treat, such as future can be used in treatment or delay degenerative disorders, repair
Overlying tissue damage, neomorph and regeneration；Or the aforementioned adipose stromal stem cell of freezen protective and establish a cell bank with
For subsequent applications, such as the research of clinic study, regenerative medicine or the development of cellular system engineering.
Above-described embodiment and attached drawing and non-limiting product form and style of the invention, any technical field it is common
The appropriate changes or modifications that technical staff does it all should be regarded as not departing from patent category of the invention.
Claims (20)
1. a kind of method of the stromal vascular confluent monolayer cells in isolated adipose tissue living cells, which is characterized in that include following step
It is rapid:
Step a provides the adipose tissue of a mankind；
The adipose tissue is placed in the closed container of a cutter device by step b；
Step c makes the cutter device bestow an active force to the closed container, enables the default cutting tool in the closed container
Fast homogeneous chopping is executed to the adipose tissue, one is obtained and homogenizes adipose tissue；
Step d homogenizes in this aforementioned shredded and a reagent is added in adipose tissue, and reaction is hydrolyzed；
Step e is filtered aforementioned, be centrifugated through processed this of the hydrolysis adipose tissue that homogenizes；And
Step f removes the supernatant after centrifuge separation, to obtain a cell precipitate, wherein cuts described in abovementioned steps b
Cutting device is a deserted homogenizer, and aforementioned deserted homogenizer includes a deserted closed container and a power list
Member, and there is a cutting tool in the deserted closed container；It is in the throwing of 15 milliliters of volumes that abovementioned steps c homogeneous, which shreds condition,
Abandoning formula closed container is built into 3 g to 6 g adipose tissues, and the revolving speed of the homogenizer be 1200 revolutions per minutes extremely
1500 revolutions per minutes, and action time is 3 minutes to 10 minutes, to carry out fast homogeneous chopping.
2. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as described in claim 1, it is characterised in that:
It may include aforementioned adipose tissue is cleaned with phosphate buffer solution the step of before step a.
3. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as described in claim 1, it is characterised in that:
Step c homogeneous shreds preferable condition and is built into 3 g of adipose tissues for the deserted closed container in 15 milliliters of volumes, carries out fast
Fast homogeneous chopping.
4. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as described in claim 1, it is characterised in that:
Reagent described in abovementioned steps d is selected from a trypsase (Trypsin), a neutral proteinase (Dispase), a glue enzyme
(Gelatase), a sodium hyaluronate enzyme (Hyaluronidase), one first collagen type enzyme (Collagenase type I) or
One or a combination set of one the 4th collagen type enzyme (Collagenase type IV) constituted group.
5. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as described in claim 1, it is characterised in that:
Hydrolysis described in abovementioned steps d is that whole process is placed in a constant temperature hybridization reaction baking oven and carries out, and condition be four degrees celsius extremely
45 degree, revolving speed be 5 revolutions per minutes to 50 revolutions per minutes and the time 0.5 hour to 24 hours.
6. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as claimed in claim 5, it is characterised in that:
Condition be it is 37 degree Celsius, revolving speed is 15 revolutions per minutes and the time 8 hours.
7. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as described in claim 1, it is characterised in that:
The condition of centrifugation described in abovementioned steps e is 400 × g, the time 10 minutes.
8. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as described in claim 1, it is characterised in that:
Abovementioned steps e is further included will filter out adipose tissue disintegrating slag after gained one filtered fluid move into a centrifuge tube the step of, with carry out from
Heart separation.
9. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as described in claim 1, it is characterised in that:
Cell precipitate described in abovementioned steps f is a stromal vascular confluent monolayer cells.
10. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as described in claim 1, feature exist
In: a step g is further included after abovementioned steps f, phosphate buffer solution is added in aforementioned cells sediment, clean aforementioned cells
Sediment, the cell precipitate being centrifuged after obtaining a cleaning to remove the supernatant containing watery blood and aforementioned agents again.
11. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as claimed in claim 10, feature exist
In: a step h is further included after abovementioned steps g to be cultivated in cell precipitate one culture medium of merging after aforementioned cleaning, to obtain
Take the cell of an amplification.
12. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as claimed in claim 11, feature exist
In: the culture medium includes: a basal medium, a serum additive, a second type fiber mother cell growth factor.
13. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as claimed in claim 12, feature exist
A fetal calf serum in: the serum additive, and the concentration expressed in percentage by volume used is percent 2 to percent 10, and this second
Fiber type mother cell growth factor is 1 ng/ml to 20 ngs/ml using concentration.
14. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as claimed in claim 11, feature exist
In: the cell of the amplification is the undifferentiated adipose stromal stem cell of essence.
15. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as claimed in claim 14, feature exist
In: the adipose stromal stem cell is at least to show cell surface antigen CD90 or CD105 or combinations thereof, and do not show CD45
Cell mass.
16. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as claimed in claim 14, feature exist
In: it later include the adipose stromal stem cell of the aforementioned amplification of step i` execution induction differentiation in abovementioned steps h, to obtain one certainly
The cell that the adipose stromal stem cell of aforementioned amplification is broken up.
17. the method for the stromal vascular confluent monolayer cells in isolated adipose tissue living cells as claimed in claim 16, feature exist
In: one of the cell, including osteocyte, fat cell or cartilage cell broken up from the adipose stromal stem cell of aforementioned amplification.
18. a kind of adipose stromal stem cell is in preparation for one of cell therapy, regenerative medicine or cellular system engineering or its group
The purposes of the medical composition of conjunction, which is characterized in that the adipose stromal stem cell is by method of claim 15
The adipose stromal stem cell of acquisition.
19. a kind of purposes of medical composition of adipose stromal stem cell in preparation for further applying, feature exist
In the adipose stromal stem cell is the adipose stromal stem cell of the amplification obtained by method of claim 14 in preceding
Stating step h includes later a step i freezen protective.
20. a kind of adipose stromal stem cell is in preparation for treating degenerative disorders, repair tissue damage, neomorph or tissue
The purposes of the medical composition of one or a combination set of regeneration, which is characterized in that the adipose stromal stem cell is wanted by right
The undifferentiated adipose stromal stem cell of essence that method described in asking 14 obtains.
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