CN106978396A - A kind of amplification cultivation method of fat mesenchymal stem cell clone - Google Patents
A kind of amplification cultivation method of fat mesenchymal stem cell clone Download PDFInfo
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Abstract
The invention discloses the amplification cultivation method of fat mesenchymal stem cell clone a kind of, being separately cultured for the extraction, the preparation of (2) special complete medium, (3) primary fat mesenchymal stem cell that method passes through (1) rich platelet Fibrin Glue is completed with four steps of biological property after Secondary Culture and the amplification of (4) different type fat mesenchymal stem cell.The present invention obtains a kind of CD54 (+) fat mesenchymal stem cell group of high-purity, this group of cell have stronger clonality, self-renewal capacity and into fat differentiation potential, apply to adipose tissue regeneration to obtain the substantial amounts of seed cell with dryness in vitro and provide new method, a kind of new therapeutic strategy is provided to improving clinically soft tissue healing or congenital body surface organ soft tissue defect deformity, meanwhile, also to ensure that its future provides sufficient theoretical foundation in the security of clinical practice.
Description
Technical field
The invention belongs to field of biomedicine technology, specifically a kind of amplification cultivation side of fat mesenchymal stem cell clone
Method.It is related to the efficient amplification cultural method and related biological Function Identification of a kind of human adipose's mescenchymal stem cell clone.
Background technology
Since Zuk in 2001 etc. has had found in adipose tissue since the mescenchymal stem cell containing multi-lineage potential first,
Fat mesenchymal stem cell has just been increasingly becoming one of most promising seed cell in adult stem cell.Adipose tissue passes through fat
Fat aspiration or surgical operation are easily obtained, and the wound to donor is relatively small, and are not related to ethics problem, as cell
Ideal tools in treatment and organizational project application, many report confirmations existing at present, fat mesenchymal stem cell is soft in skin
Tissue defect, bone and cartilage damage, central nervous system disease, hepatic sclerosis and autoimmune disease etc. have wide face
Bed application prospect [Philips BJ, Marra KG, Rubin JP.Healing of grafted adipose tissue:
current clinical applications of adipose-derived stem cells for breast and
face reconstruction.Wound Repair Regen,2014,22Suppl 1:11-3.Guillaume-Jugnot
P,Daumas A,Magalon J,Sautereau N,Veran J,Magalon G,et al.Autologous fat graft
and adipose tissue-derived stromal vascular fraction injection for hand
therapy in systemic sclerosis patients.2016,Curr Res Transl Med,64(1):35-
42.Tissiani LA,Alonso N.A Prospective and Controlled Clinical Trial on
Stromal Vascular Fraction Enriched Fat Grafts in Secondary Breast
Reconstruction.Stem Cells Int,2016,2016:2636454.].However, facing by fat mesenchymal stem cell
Bed application conversion process in, it has been found that with conventional cultivation technique be extremely hard to obtain it is substantial amounts of can keep stem cell stably
The fat mesenchymal stem cell of " dryness " for clinic need [Bunnell BA, Flaat M, Gagliardi C, Patel B,
Ripoll C.Adipose-derived stem cells:isolation,expansion
anddifferentiation.Methods,2008,45(2):115-20.Cheng NC,Chen SY,Li JR,Young
TH.Short-term spheroid formation enhances the regenerative capacity of
adipose-derived stem cells by promoting stemness,angiogenesis,and
chemotaxis.Stem Cells Transl Med,2013,2(8):584-94.].According to what is obtained at present from zoopery
Data, the minimum dose therapeutically effective of adult of mescenchymal stem cell is in (1.0-2.0) × 106/ kg's, i.e. an Individual Quality 75kg
Adult about needs to be transfused (0.75-1.5) × 108Individual cell, moreover, in order to maintain stable curative effect, it is often necessary to be directed to
Same illness carries out interim multiple infusion of therapeutic [Murphy MB, Moncivais K, Caplan AI.Mesenchymal
stem cells:environmentally responsive therapeutics for regenerative
medicine.Exp Mol Med,2013；45:e54.Fernández Vallone VB,Romaniuk MA,Choi H,
Labovsky V,Otaegui J,Chasseing NA.Mesenchymal stem cells and their use in
therapy:what has been achievedDifferentiation,2013,85(1-2):1-10.Doorn J,Moll
G,Le Blanc K,et al.Therapeutic applications of mesenchymal stromal cells:
paracrine effects and potential improvements.Tissue Eng Part B Rev,2012；18
(2):101-115.].Therefore need to set up the cultured and amplified in vitro system of a stability and high efficiency, make limited initial cell through expansion
Enough cell quantities can be reached after increasing and there is specific differentiation function to meet the demand of clinic.How to expand in vitro
Mescenchymal stem cell is harvested in journey to greatest extent, while the physiological property for keeping stem cell is that clinical cytology laboratory suddenly waits to solve
Key issue certainly.
In conventional medium, mescenchymal stem cell easily loses self-renewal capacity, easily aging, or " spontaneity " point
Turn to Gegenbaur's cell, fat cell or fibroblast [Mark P, Kleinsorge M, Gaebel R, Lux CA, Toelk
A,Pittermann E,David R,Steinhoff G,Ma N.Human Mesenchymal Stem Cells Display
Reduced Expression of CD105after Culture in Serum-Free Medium.Stem Cells Int,
2013；2013:698076.Tsang WP,Shu Y,Kwok PL,Zhang F,Lee KK,Tang MK,Li G,Chan KM,
Chan WY,Wan C.CD146+human umbilical cord perivascular cells maintain stemness
under hypoxia and as a cell source for skeletal regeneration.PLoS One,2013,8
(10):e76153.Zhang D,Kilian KA.The effect of mesenchymal stem cell shape on
the maintenance of multipotency.Biomaterials,2013,34(16):3962-9.]；It is substantial amounts of at present
Research confirms, stem cell in vitro in amplification procedure " dryness " forfeiture prompting, maintain mesenchyma dry thin in microenvironment in vivo
Caused by a variety of key factors of born of the same parents are lacked in traditional cultivating system, [Scadden DT.The stem-cell niche as
an entity of action.Nature,2006,441(7097):1075-9.Sharma MB,Limaye LS,Kale
VP.Mimicking the functional hematopoietic stem cell niche in vitro:
recapitulation of marrow physiology by hydrogel-based three-dimensional
cultures of mesenchymal stromal cells.Haematologica,2012,97(5):651-60.]；Therefore,
A variety of required cell factors that enough contents are provided in cultivating system keep dry to the external reconstruction of cell micro-environment with effective
What cell characteristics were likely necessary.
Since two thousand five, our research team builds using the subcutaneous fat at mankind's different anatomic position to be tissue-derived
Oneself a whole set of experimental technique platform and method have been found, the side of being separately cultured of fat mesenchymal stem cell is further optimized
Case, and pluripotent differentiation phenotypic evaluation is carried out to it, grown by optimizing it in cell injuring model or internal migration process
The biological functions such as propagation, Multidirectional Differentiation, paracrine, achieve gratifying achievement in research, are fat mesenchymal stem cell application
Promote in organizational project, regenerative medicine and clinically wound multiple, regeneration, organ to rebuild to provide reliable theoretical foundation.
The content of the invention
It is an object of the invention to provide the amplification cultivation method of fat mesenchymal stem cell clone a kind of.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
A kind of amplification cultivation method of fat mesenchymal stem cell clone, comprises the following steps：
(1) extraction of rich platelet Fibrin Glue
50~100ml of patient venous blood is extracted, sterile negative pressure is sub-packed in and tries intravascular, using rotating speed 2700rpm/min
Supercentrifuge is centrifuged, and centrifugation time is 12min, extracts the spawn rich in cell factor, stands 5min, you can obtain
Rich platelet fibrin gel, now blood points 3 layers：The superiors are platelet poor plasmas, are pale yellow transparent clear liquid；
Intermediate layer is rich platelet fibrin, is light yellow gel shape material (as shown in Figure 1)；Orlop is bib, is
Dark red color substance；Wherein unnecessary red blood cell is discharged, rich platelet fibrin and platelet poor plasma are added containing body together
The DMEM culture mediums of fraction 100U/mL penicillin, 100 μ g/mL streptomysins, it is standby to be positioned over 4 DEG C of refrigerator storages, is used after 7d
The concentration of various cell factors show in EUSA detection culture medium, VEGF, alkaline into fibre
Tie up Porcine HGF, platelet derived growth factor, transforming growth factor-beta 1, EGF, interleukin 4,
The peak concentration of interleukin-6, Fibroblast collagenase and insulin-like growth factor-i 7d after extraction, afterwards
Higher cytokine levels (as shown in Figure 2) are still kept in slow decline, but during storage 28d.
(2) preparation of special complete medium
The rich platelet fibrin and Platelet poor blood of DMEM high glucose medium 500ml+100ml venous blood separation and Extractions
+ 1% penicillin, streptomysin are starched, 4 DEG C save backup.
(3) primary fat mesenchymal stem cell is separately cultured and Secondary Culture
Outside or lower abdomen lower negative pressure extract adipose tissue 50ml in patient thigh's epimere, use rotating speed for 600rpm/
Min centrifuges, centrifugation time is 2min, with 0.1% NTx enzyme 40~50min of constant temperature digestion, with step (2) etc.
The special complete medium of amount uses rotating speed to be 5min for 1300rpm/min centrifuges, centrifugation time after neutralizing, and goes removal of impurities
Matter and supernatant, with 200 mesh nylon net filters after precipitation suspension, then with special complete medium by cell suspension, by 1 × 105/ml
It is inoculated in 25cm2Blake bottle in, be placed in volumetric concentration for 5%CO2Middle saturated humidity, in 37 DEG C of constant incubators carry out individual layers
Nutrient solution is changed after cell culture, 24~48h once, and later every 3~4d changes liquid 1 time and dynamically observes the growth characteristics of cell, 7
The fusion of~10d cell growths is up to after 80%~90%, and now cell is referred to as primary fat mesenchymal stem cell and is designated as ASCs-P0,
Digested through 0.25% pancreatin, Secondary Culture is carried out by 1: 3, first generation cell is designated as ASCs-P1.Choose well-grown ASCs-
It is positive thin that P1 carries out Flow cytometry CD29, CD44, CD45, CD49d, CD54, CD105, CD106 surface molecular for cell
Born of the same parents' number, while being sorted ASCs-P1 with immunological magnetic bead sorting method, obtains CD54 (+) ASCs-P1, by the CD54 (+) of acquisition
ASCs-P1 is with special complete medium culture and carries out continuing to pass on amplification, by CD54 (+) ASCs-P3, CD54 (+) ASCs-
P10 forms compound with collagen protein sponge support respectively, and in vitro culture takes out material to 7d in adipogenic induction culture medium,
Fixed with 2.5% glutaraldehyde, with gradient alcohol dehydration, critical point drying, cross-sectional surface metal spraying, scanning electron microscopic observation；Choose
Well-grown CD54 (+) ASCs-P3, CD54 (+) ASCs-P10 is for cell and CD54 (-) ASCs-P3, CD54 (-) ASCs-
P10 carries out routine into fat induction culture 14d, compares the differentiation into adipocytes in vitro of each group cell.
(4) biological property after the amplification of different type fat mesenchymal stem cell
Primary fat mesenchymal stem cell is adherent in 24h after inoculation, and the form of cell is short fusiformis or triangle, in spy
In different complete medium incubation, cloning growth is presented in fat mesenchymal stem cell, and 7~10d number of cell clones is rapid
Increase and form fine and close cell mass, cell is arranged in directionality, still cloning is presented in visible fat mescenchymal stem cell after passage
Mushroom out and be uniformly distributed；Fat mesenchymal stem cell growth conditions in special complete medium incubation are lived very much
Jump, during the 10th generation amplification in vitro, multiplication rate is basicly stable, and cellular morphology is basic in spindle shape, growth course
Keep constant, the 10th fat subsitutes mescenchymal stem cell still has vigorous multiplication capacity (as shown in Figure 3), test result indicates that,
Using special complete medium, the fat mesenchymal stem cell of materials 20ml adipose tissue separation passes through the external expansion of 3 weeks or so
Increase, sum can reach 1 × 108More than；1st generation fat mesenchymal stem cell, can without adipogenic induction 14d before immunomagnetic beads screening
See it is intracellular there are bright fat drips to be formed, the visible fat drips of oil red O stain are dyed to cerise；After osteogenic induction 21d, it is seen that typical case
Mineralising calcium tubercle formed, Alizarin red staining be in cerise；Into after chondrocyte induction 14d, it is seen that the cell of high aggregation sample growth
Group, in patch shape or nodositas, peripheral cell is radial, and tubercle is larger, naked eyes it is visible, alcian blue dyeing prompting tubercle and
The cell of periphery aggregation is in blue (as shown in Figure 4)；1st generation fat mesenchymal stem cell carries out Flow cytometry result and shown
It is positive expression to show CD29, CD44, CD49d, CD54, CD105 surface molecular, and CD45 and CD106 be feminine gender (such as Fig. 5 institutes
Show)；After CD54 (+) ASCs-P3, CD54 (+) ASCs-P10 adipogenic induction cultures 7d, observed under ESEM between display, fat
Mesenchymal stem cells stretch on timbering material surface and internal hole wall, sticked, and cell surface has fat drips attachment, and cellular morphology is generally inclined
Circular, spindle shape or it is irregular spread out, cell quantity gradually increases, and is distributed in aggregation, and cell week is with a large amount of matrix, says
Bright collagen scaffold has very good biocompatibility with cell, and pole is beneficial to the growing multiplication (as shown in Figure 6) of cell；CD54
After (+) ASCs-P3, CD54 (+) ASCs-P10 fat mesenchymal stem cells adipogenic induction starts, cell proliferation rate slows down, carefully
Cell space product becomes larger, and occurs the vacuole differed in size and fat drips, and progressively mutually fusion in 7d or so endochylemas, forms larger fat
Drop；When inducing 14d, cell differentiation peaks, and intracellular fat drips are after oil red O stain, shown in red drop, and the two is in shape
Without marked difference in state (as shown in Fig. 7 A, B)；And after CD54 (-) ASCs-P3, CD54 (-) ASCs-P10 adipogenic inductions, cell
In polygon, and only there is red dye liquor drop, forms distinct right with the former in bulky, form generation change in a few cell
Than (as shown in Fig. 7 C, D)；Two groups of mature fat cell density of CD54 (+) ASCs-P3, CD54 (+) ASCs-P10 are (such as Fig. 8 institutes
Show), intracellular fat drips content detection (as shown in Figure 9), into lipid phase correlation gene (as shown in Figure 10) and protein expression (such as Figure 11 institutes
Show) be all remarkably higher than two groups of CD54 (-) ASCs-P3, CD54 (-) ASCs-P10, and These parameters CD54 (+) ASCs-P3,
Between two groups of CD54 (+) ASCs-P10 and CD54 (-) ASCs-P3, CD54 (-) ASCs-P10 is without marked difference.
Beneficial effects of the present invention
The present invention obtains a kind of CD54 (+) fat mesenchymal stem cell group of high-purity, and this group of cells have stronger
Clonality, self-renewal capacity and into fat differentiation potential, continuously reaching for the 10th generation is difficult aging, still maintains preferably
" dryness " feature, for obtain in vitro the substantial amounts of seed cell with dryness apply to adipose tissue regeneration provide it is new
Method, a kind of new treatment is provided to improving clinically soft tissue healing or congenital body surface organ soft tissue defect deformity
Strategy, meanwhile, also to ensure that its future provides sufficient theoretical foundation in the security of clinical practice.
Brief description of the drawings
Fig. 1 is the extraction figure rich in cell factor Fibrin Glue of the present invention.
Fig. 2 is the different time points rich platelet Fibrin Glue release cell factor MBP enzyme linked immuno-adsorbent assay of the present invention
As a result.
Fig. 3 is aspect graph under the primary of the present invention, 1st generation, the 3rd generation, the 10th fat subsitutes mescenchymal stem cell microscope.
In figure, (A) is that primary, (B) is that 1st generation, (C) are that the 3rd generation, (D) were the 10th generation.
Fig. 4 is 1st generation fat mesenchymal stem cell Multidirectional Differentiation experimental result picture of the present invention.
In figure, A is into fat, B skeletonization, C into cartilage.
Fig. 5 is 1st generation fat mesenchymal stem cell streaming art detection surface C D molecular results of the present invention
Fig. 6 is the present invention into after fat induction 7d, cellular morphology figure under ESEM.
In figure, A is that the generation of CD54 (+) fat mesenchymal stem cell-the 3, B are CD54 (+) fat mesenchymal stem cell-the 10
Generation.
Fig. 7 is each group cell of the present invention into oil red O stain result after fat induction 2 weeks
In figure, A is that the generation of CD54 (+) fat mesenchymal stem cell-the 3, B are CD54 (+) fat mesenchymal stem cell-the 10
Generation, C are that the generation of CD54 (-) fat mesenchymal stem cell-the 3, D are the generation of CD54 (-) fat mesenchymal stem cell-the 10.
Fig. 8 be each group fat mesenchymal stem cell of the present invention into fat cell density ratio after fat induction 2 weeks compared with.
Fig. 9 is each group fat mesenchymal stem cell of the present invention into intracellular fat drips comparision contents after fat induction 2 weeks.
Figure 10 be each group fat mesenchymal stem cell of the present invention into after fat induction 2 weeks into fat related gene expression ratio
Compared with.
Figure 11 be each group fat mesenchymal stem cell of the present invention into after fat induction 2 weeks into fat correlative protein expression ratio
Compared with.
Figure 12 is transplantation effect after different type fat stem cell of the present invention is mixed with collagen sponge scaffold
In figure, a is graft cambium general condition after being implanted into 12 weeks, and b is two groups of graft cambium weight in wet base ratios
Compared with A groups are that compound CD54 (+) ASCs-P3, the B groups of collagen scaffold are that collagen scaffold is combined CD54 (-) ASCs-P3, and c is the HE of A groups
Dyeing, d dyes for the HE of B groups；
Figure 13 is mature fat cell density and intracellular fat drips comparision contents in two groups of cambiums of the invention.
In figure, a is fat cell density, and b is intracellular fat drips content；A groups are that collagen scaffold is combined CD54 (+) ASCs-
P3, B group are that collagen scaffold is combined CD54 (-) ASCs-P3.
Embodiment
With reference to embodiment, the invention will be further described.
Embodiment 1
Transplanting and detection after different type fat stem cell is mixed with collagen sponge scaffold, operating procedure are as follows：
1. the preparation of cell-scaffold compound (graft)
Sterile collagen protein stent material is tailored into 10mm × 10mm × 5mm sizes, the 3rd fat subsitutes of culture are dry thin
Cell suspension is made after born of the same parents' digestion, every group of cell density is 1 × 107/ ml, cell suspensions of the 1ml after labeled is mixed with support
Close, stand after 4h, add complete culture solution, 37 DEG C, 5%CO2And overnight incubation in the incubator of saturated humidity, the shifting of morning next day
Plant in nude mouse.
2. test animal and packet
2 groups of experiment point：Nude mice 10, weight 20g ± 5g, male and female are regardless of.
A groups：Collagen scaffold is combined in CD54 (+) ASCs-P3, implantation nude mice left rear side dorsal sc muscular fascia.
B groups：Collagen scaffold is combined in CD54 (-) ASCs-P3, implantation nude mice right lateral side dorsal sc muscular fascia.
3. postoperative materials
Graft is implanted into 12 weeks after operation, and graft is taken out in operation again, visually observes the form and collagen scaffold of graft
Residual condition, has seen whether cambium generation；Carefully cambium is stripped down, new life is weighed up with electronic balance immediately
The weight in wet base of thing, is then divided into two parts by tissue block, and portion does oil red O stain for frozen section；Another is with 4% poly first
Aldehyde is fixed, dehydration of alcohol, and FFPE, wax stone is marked respectively, for doing HE dyeing and immunofluorescence dyeing.
4. detection project
Histological examination：Paraffin specimen section conventional H E dyeing and frozen section oil red O stain, fluorescence microscopy Microscopic observation
The cell component of section cambium, compares each group mature fat cell density, intracellular fat drips content；
5. transplanting result in body
A group graft cambium weight in wet bases (as shown in figure 12), mature fat cell density (as depicted in fig. 13 a), cell
Interior fat drips content (as illustrated in fig. 13b) is high compared with B groups, and statistical analysis difference has significant, these results indicate that through this
After fat mesenchymal stem cell CD54 (+) ASCs of invention amplification cultivation is combined with collagen scaffold, break up after implanting into fat
Efficiency is significantly improved, and is conducive to building large volume of engineered fat.
Claims (1)
1. a kind of amplification cultivation method of fat mesenchymal stem cell clone, it is characterised in that comprise the following steps：
(1) extraction of rich platelet Fibrin Glue
50~100ml of patient venous blood is extracted, sterile negative pressure is sub-packed in and tries intravascular, using rotating speed 2700rpm/min at a high speed
Centrifuge, centrifugation time is 12min, extracts the spawn rich in cell factor, stands 5min, you can obtain rich blood
Platelet fibrin gel, now blood points 3 layers：The superiors are platelet poor plasmas, are pale yellow transparent clear liquid；It is middle
Layer is rich platelet fibrin, is light yellow gel shape material；Orlop is bib, is dark red color substance；Discharge
Wherein unnecessary red blood cell, adds 100U/mL containing volume fraction blue or green together by rich platelet fibrin and platelet poor plasma
In mycin, the DMEM culture mediums of 100 μ g/mL streptomysins, it is standby to be positioned over 4 DEG C of refrigerator storages, and Enzyme-linked Immunosorbent Assay is used after 7d
The concentration of various cell factors is shown in testing inspection culture medium, VEGF, basic fibroblast growth because
Son, platelet derived growth factor, transforming growth factor-beta 1, EGF, interleukin 4, interleukin-6,
The peak concentration of Fibroblast collagenase and insulin-like growth factor-i 7d after extraction, afterwards in slow decline, but
Higher cytokine levels are still kept during storage 28d；
(2) preparation of special complete medium
The rich platelet fibrin and platelet poor plasma of DMEM high glucose medium 500ml+100ml venous blood separation and Extractions+
1% penicillin, streptomysin, 4 DEG C save backup；
(3) primary fat mesenchymal stem cell is separately cultured and Secondary Culture
Outside or lower abdomen in patient thigh's epimere, extract adipose tissue 50ml using lower negative pressure, use rotating speed for 600rpm/
Min centrifuges, centrifugation time is 2min, with 0.1% NTx enzyme 40~50min of constant temperature digestion, with step (2) etc.
The special complete medium of amount uses rotating speed to be 5min for 1300rpm/min centrifuges, centrifugation time after neutralizing, and goes removal of impurities
Matter and supernatant, with 200 mesh nylon net filters after precipitation suspension, then with special complete medium by cell suspension, by 1 × 105/ml
It is inoculated in 25cm2Blake bottle in, be placed in volumetric concentration for 5%CO2Middle saturated humidity, in 37 DEG C of constant incubators carry out individual layers
Nutrient solution is changed after cell culture, 24~48h once, and later every 3~4d changes liquid 1 time and dynamically observes the growth characteristics of cell, 7
The fusion of~10d cell growths is up to after 80%~90%, and now cell is referred to as primary fat mesenchymal stem cell and is designated as ASCs-P0,
Digested through 0.25% pancreatin, Secondary Culture is carried out by 1: 3, first generation cell is designated as ASCs-P1.Choose well-grown ASCs-
It is positive thin that P1 carries out Flow cytometry CD29, CD44, CD45, CD49d, CD54, CD105, CD106 surface molecular for cell
Born of the same parents' number, while being sorted ASCs-P1 with immunological magnetic bead sorting method, obtains CD54 (+) ASCs-P1, by the CD54 (+) of acquisition
ASCs-P1 is with special complete medium culture and carries out continuing to pass on amplification, by CD54 (+) ASCs-P3, CD54 (+) ASCs-
P10 forms compound with collagen protein sponge support respectively, and in vitro culture takes out material to 7d in adipogenic induction culture medium,
Fixed with 2.5% glutaraldehyde, with gradient alcohol dehydration, critical point drying, cross-sectional surface metal spraying, scanning electron microscopic observation；Choose
Well-grown CD54 (+) ASCs-P3, CD54 (+) ASCs-P10 is for cell and CD54 (-) ASCs-P3, CD54 (-) ASCs-
P10 carries out routine into fat induction culture 14d, compares the differentiation into adipocytes in vitro of each group cell；
(4) biological property after the amplification of different type fat mesenchymal stem cell
Primary fat mesenchymal stem cell is adherent in 24h after inoculation, and the form of cell is short fusiformis or triangle, special complete
During full medium culture, cloning growth is presented in fat mesenchymal stem cell, and 7~10d number of cell clones increases rapidly
And fine and close cell mass is formed, cell is arranged in directionality, and still visible fat mescenchymal stem cell presentation cloning is rapid after passage
Grow and be uniformly distributed；Fat mesenchymal stem cell growth conditions in special complete medium incubation are very active,
During 10th generation amplification in vitro, multiplication rate is basicly stable, and cellular morphology is basic in spindle shape, growth course keeps not
Become, the 10th fat subsitutes mescenchymal stem cell still has vigorous multiplication capacity, test result indicates that, use special complete culture
Base, the fat mesenchymal stem cell of materials 20ml adipose tissue separation passes through the amplification in vitro of 3 weeks or so, and TCS can reach
1×108More than；1st generation fat mesenchymal stem cell is without adipogenic induction 14d before immunomagnetic beads screening, it is seen that intracellular to have
Bright fat drips are formed, and the visible fat drips of oil red O stain are dyed to cerise；After osteogenic induction 21d, it is seen that typical mineralising calcium tubercle
Formed, Alizarin red staining is in cerise；Into after chondrocyte induction 14d, it is seen that the cell mass of high aggregation sample growth, in patch shape or
Nodositas, peripheral cell is radial, and tubercle is larger, naked eyes it is visible, alcian blue dyeing prompting tubercle and periphery aggregation it is thin
Born of the same parents are in blueness；1st generation fat mesenchymal stem cell carry out Flow cytometry result show CD29, CD44, CD49d, CD54,
CD105 surface moleculars are positive expression, and CD45 and CD106 is negative as shown in table 1；CD54(+)ASCs-P3、CD54(+)
After ASCs-P10 adipogenic induction cultures 7d, observe display under ESEM, fat mesenchymal stem cell on timbering material surface and
Internal hole wall stretches, sticked, and cell surface has a fat drips attachment, cellular morphology be generally partially circular, spindle shape or it is irregular spread out,
Cell quantity gradually increases, and is distributed in aggregation, and cell week is with a large amount of matrix, illustrates that collagen scaffold and cell have very good
Good biocompatibility, pole is beneficial to the growing multiplication of cell；CD54 (+) ASCs-P3, CD54 (+) ASCs-P10 fat mesenchymals
After stem cell adipogenic induction starts, cell proliferation rate slows down, and cell volume becomes larger, and occurs size in 7d or so endochylemas not
Deng vacuole and fat drips, and progressively mutually fusion, form larger fat drips；When inducing 14d, cell differentiation peaks, intracellular
Fat drips are after oil red O stain, shown in red drop, and the two is morphologically without marked difference；And CD54 (-) ASCs-P3, CD54
After (-) ASCs-P10 adipogenic inductions, cell volume is huge, form generation change, in polygon, and only occurs in a few cell
Red dye liquor drop, sharp contrast is formed with the former；Two groups of mature fat cells of CD54 (+) ASCs-P3, CD54 (+) ASCs-P10 are close
Degree, intracellular fat drips content detection, CD54 (-) ASCs-P3, CD54 (-) is all remarkably higher than into lipid phase correlation gene and protein expression
Two groups of ASCs-P10.
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| WO2019144971A1 (en) * | 2018-01-29 | 2019-08-01 | 中国科学院上海生命科学研究院 | Icam-1 marker and application thereof |
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| CN109825471A (en) * | 2019-03-19 | 2019-05-31 | 杨姣姣 | A kind of external efficient amplification agent of fat mesenchymal stem cell, amplification method |
| CN109825471B (en) * | 2019-03-19 | 2022-08-19 | 上海揽微赛尔生物科技有限公司 | Adipose-derived mesenchymal stem cell in-vitro efficient amplification agent and amplification method |
| CN110272865A (en) * | 2019-07-17 | 2019-09-24 | 中国医科大学附属第一医院 | A kind of method of separating-purifying mesenchymal stem cell subgroup |
| CN117603904A (en) * | 2022-09-28 | 2024-02-27 | 昆明学院 | Adipocyte sphere and preparation method and application thereof |
| CN117603904B (en) * | 2022-09-28 | 2024-04-23 | 昆明学院 | Adipocyte sphere and preparation method and application thereof |
| CN117660318A (en) * | 2024-02-01 | 2024-03-08 | 广东赛尔生物科技有限公司 | Method for in-vitro amplification of adipose-derived stem cells and SVF and application of combination of method for cosmetic filling |
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