CN107224614A - A kind of preparation and its clinical practice of the Adipose-derived stromal cells cograft material rich in cell factor - Google Patents
A kind of preparation and its clinical practice of the Adipose-derived stromal cells cograft material rich in cell factor Download PDFInfo
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Abstract
The invention discloses a kind of preparation and its clinical practice of the Adipose-derived stromal cells cograft material rich in cell factor.Acquisition and purifying that preparation method is rolled into a ball by 1) lipochondrion；2) acquisition of nanometer adipose stromal cells；3) biological characteristics of nanometer adipose-derived stem cells (NFSCs) and Multidirectional Differentiation identification；4) four steps of acquisition rich in cell factor Fibrin Glue prepare graft materials, reparation and correction for clinical upper surface portion, chest and other body surface soft tissue depressions deformity.The present invention promotes wound or wound repair based on stromal vascular fraction and reduces the new technology that scar proliferation, promotion organization Regeneration and Repair and organ are rebuild, shorten the rehabilitation duration of patient, improve Quality of rehabilitation, good curative effect is obtained, the autologous adipose tissue skin grafing and mending soft tissue or organ defect, burn wound, refractory wounds and Mammary cancer breasst reconstruction aided in for clinically cell provides experimental basis and theories integration.
Description
Technical field
The invention belongs to field of biomedicine technology, specifically a kind of Adipose-derived stromal cells rich in cell factor are answered
Close the preparation and its clinical practice of graft materials.
Background technology
Due to body surface organization's organ depressed deformity that wound, infection, tumor resection or birth defects reason etc. are caused, remove
Outside influence appearance, the mental health of influence patient that can also be in various degree and social communication activity.Autologous fat transplantation is filled out
It is current treatment one of body surface soft tissue depression or the asymmetric main method of profile to fill.Its major defect is easily inhaled after transplanting
Receive, absorptivity accounts for 20%~90%, need to receive to be repeated several times to inject mostly to can be only achieved more satisfied effect, therefore
The absorptivity after fat transfer how is reduced, survival rate is improved as study hotspot in recent years.
Fat cell lacks effective blood supply in transplanting early stage, before the blood supply of graft is fully set up, the fat of transplanting
The Major Nutrient of tissue derive from surrounding tissue liquid infiltration, and the adipose tissue of middle body often because with surrounding tissue away from
From larger, hypoxic-ischemic overlong time, it is easier to necrose, liquefy.Brazilian Carpaned doctor just proposed " side in 1993
The concept of margin zone " simultaneously it was observed that there are about 40% tissue survival in the edge within graft edge about 1.5 ± 0.5mm
Area, has scholar's research to find that graft central area fibrosis is more compared with marginal zone, fat cell small volume illustrates graft
Absorption occurs mostly in middle section, therefore improves the nutritional status of graft central area early stage, promotes blood supply to set up to pass
Important [Carpaneda CA, Ribeiro MT.Study of the histologic alterations and
viability of the adipose graft in humans.AestheticPlast Surg, 1993,17(1):43-
47.Doi K,Ogata F,Eto H,Kato H,Kuno S,Kinoshita K, Kanayama K,Feng J,Manabe I,
Yoshimura K.Differential contributions of graft-derived and host-derived
cells in tissue regeneration/remodeling after fat grafting.Plast Reconstr
Surg,2015,135(6):1607-1617.]。
Separated first from adipose tissue from Zuk in 2001 etc., extract fat stem cell (adipose-derived
Stem cells, ASCs) and since naming, ASCs because wide material sources, convenient material drawing, tissue damage are small, expand rapid the advantages of
The ideal tools in cell therapy and organizational project application are turned into.Numerous studies show, ASCs injury repairs, anti-ageing in vivo
Aspect of waiting for a long time can play more obvious action；A realization part for its biological action passes through Multidirectional Differentiation ability, ASCs
The various kinds of cell such as fat, bone, cartilage, muscle, nerve can be divided into, supplement substitutes aging, the histocyte of defect；Another part
By paracrine function, ASCs can secrete cytokine profiles, polypeptide isoreactivity composition, improve damaged cell vitality and
Anti-apoptotic ability.ASCs abundances, can be divided into a variety of pedigrees, can secrete cytokine profiles, and make with immunological regulation
With its outstanding behaviours in regeneration field makes it have wide potential applicability in clinical practice.[Zuk PA,Zhu M,
Mizuno H,Huang J,Futrell JW,Katz AJ,Benhaim P,Lorenz HP,Hedrick
MH.Multilineage cells from human adipose tissue:implications for cell-based
therapies.Tissue Eng,2001,7(2):211-28.Uzbas F,May ID,Parisi AM,Thompson SK,
Kaya A,Perkins AD,Memili E.Molecular physiognomies and applications
ofadipose-derived stem cells.Stem Cell Rev,2015,11(2):298-308.]
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 are found, further optimization ASCs is separately cultured scheme, and it is dived more
Energy phenotypic differentiation identification, passes through and ginsenoside Rg1 or autologous PRP/PRF is added in cell injuring model or internal migration process
Deng composition, observation and the situation of change for evaluating cell phenotype and other biological behaviours, while it is dry to fat to evaluate these compositions
The influence that growth and proliferation of cell, Multidirectional Differentiation, paracrine function and particular organization build, achieves gratifying achievement in research, is
Fat stem cell is applied to organizational project, regenerative medicine research and clinically promotes wound repair, regeneration, organ to rebuild
And cell therapy provides theoretical foundation.
The content of the invention
Present invention solves the problem in that providing a kind of Adipose-derived stromal cells cograft material rich in cell factor
The preparation and its clinical practice of material.
The technical scheme that the present invention solves above-mentioned technical problem is as follows：
1. a kind of preparation of the Adipose-derived stromal cells cograft material rich in cell factor, preparation method include with
Lower step：
1) acquisition and purifying of lipochondrion group
Outside or lower abdomen in upper lap is chosen, to supply area, to reserve air method lower negative pressure with hand-held 20ml syringes and extract,
According to patient profiles, select fat and supply after zone position, local swelling liquid infiltration anesthesia, using the porous special liposuction of designed, designed
Pin is aspirated, and is aspirated during suction from deep layer to shallow-layer, and sector is aspirated repeatedly；By the lipochondrion pumped out, using physiology
Normal saline washing is standby；Take low-speed centrifugal method to obtain and purify lipochondrion group, centrifugal rotational speed 600rpm/min, centrifugation time
2min, operating process whole process follows sterile closed operating principle, and the time of contact of adipose tissue and air is reduced as far as possible.Such as Fig. 1
It is shown.
2) acquisition of nanometer adipose stromal cells
By a part of lipochondrion roll into a ball through machinery smash to pieces emulsion process prepare rich in fat stem cell nanometer lipid substrate it is thin
Born of the same parents, by step 1) Grainy fat tissue via hole diameter 0.5mm after preliminary treatment strainer filtering, then by Grainy fat tissue through 2 20mL
Quickly air exercise 30 times of the fatty special-purpose emulsifying device of injection needle tube connection nanometer, machinery emulsification, with the filter screen in 0.5mm apertures mistake again
Filter, through rotating speed is afterwards most 8000rpm/min, and centrifugation time is that 5min high speed centrifugations remove after grease obtained fat emulsification liquid i.e.
It is that nanometer is fatty as shown in Fig. 1, the nanometer fat after the emulsification of this part carries more stromal vascular fraction and fat is dry carefully
Born of the same parents, are conducive to increasing stromal vascular fraction and the contact surface of fat cell, the survival rate of fat graft are improved, by machinery emulsification
The mescenchymal stem cell that Grainy fat tissue is separately cultured and obtained after passing on is referred to as a nanometer adipose-derived stem cells.
3) biological characteristics of nanometer adipose-derived stem cells and Multidirectional Differentiation identification
Through step 2) the nanometer adipose stromal cells after machinery emulsification contain after more stromal vascular fraction, culture 24h
The cell for beginning with a small amount of fibroblast sample form is dispersed in adherent, and most of non-attached cell is spherical or circular miscellaneous thin
Born of the same parents.Liquid is changed after 24-48h first to remove after non-attached cell, most of attached cell is thin in roomy flat fibroblast sample
Born of the same parents, with the extension of incubation time, these attached cells grow in colony shape, and colony is not of uniform size；Cellular morphology is in colony
Typical spindle cell.About 7-10d is up to 80%~90% compacted zone individual layer.Cellular morphology is mainly fusiformis after passage, is such as schemed
Shown in 2 A, B, C.Vitro growth rates are substantially speeded compared with primary cell after passage, if by 1：3 passages, 2-3d can be covered with.
Continuous passage culture 10 generation cell does not occur obvious aging phenomenon.Cell process substantially, in fusiformis or polygonal, grows after passage
Comparatively fast, cell doubling time is averagely about 40h after testing, and the fat respectively obtained for cellular morphology with traditional collagenase digestion is done
Cell is similar.3rd generation cell is through adipogenic induction 14d, it is seen that intracellular to have bright fat drips to be formed, the visible fat drips of oil red O stain
Cerise is dyed to as shown in Fig. 2 D, E.After osteogenic induction 21d, it is seen that typical mineralising calcium tubercle is formed, Alizarin red staining
Wu's cerise is as shown in Fig. 2 F, G；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；Tubercle is larger, and naked eyes are visible；It is thin that alcian blue dyeing prompting tubercle and periphery are assembled
Born of the same parents are in blueness as shown in Fig. 2 H, I.3rd generation nanometer adipose-derived stem cells flow cytometry show CD29, CD44,
CD49d, CD54, CD90 and CD105 are positive, and CD34, CD45 and CD106 are negative, as shown in Figure 5.
4) it is rich in the acquisition of cell factor Fibrin Glue
Extract in same individual appropriate venous blood, the sterile negative pressure heparin tube being placed in, using Choukroun ' s methods from
Heart rotating speed is that 2700rpm/min, centrifugation time are that high speed centrifugation of 12min extracts the spawn rich in cell factor.
At once with 20ml asepsis injectors collection Venous Blood liquid 20-40ml, and closure is sealed.Sterile centrifuge shield is put into
In centrifuge, the 20ml syringes that then will be equipped with Venous Blood liquid are put into sleeve pipe, are centrifuged by 2700rpm/min of rotating speed
12min.Stand and supernatant is abandoned after 3~5min, remove bottom bib, collect intermediate filament protein gel layer, filtering is removed
Serum, will be enriched in cell factor Fibrin Glue and collects standby, as shown in Figure 3.Simultaneously to rich in cell factor Fibrin Glue
The detection of cytokine release concentration is carried out within 1~168h periods, VEGF blood platelet source is as a result found
Property growth factor, transforming growth factor-beta 1, EGF, interleukin-6, Fibroblast collagenase and insulin
The concentration of like growth factor -1 is in rising trend, as shown in Figure 4.These cell factors have weight to promotion organization reparation and regeneration
The effect wanted.
5) clinical practice situation
Graft is by structural fatty particle, nanometer adipose stromal cells and rich in cell factor Fibrin Glue by certain
Ratio is mixed, and entry point, local anaesthesia, from remote near slow are used as by area's surrounding markings, selecting more hidden position
Slow injection graft, is uniformly distributed it.Before injection, skin first is stabbed out with sharp pin, then changes blunt needle into and injects composite graft,
Whether first pumpback has blood back, especially near the eyes, it is to avoid be injected into intravascular, during injection, gives the pressure of minimum, slow note
Enter.Interruption, sector, wire injection are should be noted that during injection, using multi-point layered injection, it is to avoid agglomerate situation occurs, and influences blood
Fortune.Injection volume should be 25%~30% or so more than aequum：Part massage after injection, makes graft be evenly distributed.It is postoperative slight
Compressing dressing fixes 2 days.Vein uses antibiotic 3 days, prevention infection.
Project of the present invention is using nanometer fat of the fresh separated rich in stromal vascular fraction, rich in cell factor fibrin
Glue is formed after composite graft with Auto fat granule, to 62 Facial Depressions, skin aging and mastatrophy or congenital hair
Educate bad patient and carry out local injection transplantation treatment, by the follow-up of 12~24 months, it is found that fatty survival condition is good, greatly
Some cases single injection can reach promising result, and only 5 (8.06%) needs 2 injections of row, and traditional autologous fat
Transplanting Most patients then need repeated multiple times injection treatment, and graft generation tumour, liquefied probability are higher.In addition institute
There is patient is postoperative not find the bad complication such as local infection, liquefaction of fat, scleroma, cacesthesia, illustrate this method safety
Property it is reliable, and effect is significantly, can as all kinds of soft tissue defects of clinical treatment and skin senescence preferred option.While we
It has also been found that the skin quality that all patients receive transplantation site has obtained different degrees of improvement, such as color spot desalination, microgroove disappear, pore
Reduce etc., its main mechanism is the paracrine function of the fatty medium vessels matrix components of nanometer and rich in cell factor fiber
Cell factor in protein adhesive has played regeneration and skin anti-aging effect.
In summary, using nanometer fat of the fresh separated rich in stromal vascular fraction, rich in cell factor fibrin
Glue is controlled with anatomic structures local solids flux as face, chest soft tissue depression or the moulding of defect patient by a certain percentage
Treat, postoperative patient satisfaction is high, adipose tissue by area's long-term surviving, can have no bad complication, and single injection can be obtained
Satisfied effect is obtained, is good security, the significant face contour of late result and chest shaping methods, this method can be notable
Fat transfer survival rate is improved, moulding, chest enlarge, finishing impression face contour is reached and the effect of facial skin quality can be improved, be worth into one
Step further investigation and popularization and application.
Beneficial effects of the present invention：
The present invention promotes wound or wound repair based on stromal vascular fraction and reduces scar proliferation, promotion organization regeneration to repair
The new technology that multiple and organ is rebuild, shortens the rehabilitation duration of patient, obtains good curative effect, is the autologous of clinically cell auxiliary
Adipose tissue transplantation repairs soft tissue or organ defect, burn wound, refractory wounds and Mammary cancer breasst reconstruction are provided
Experimental basis and theories integration.
Brief description of the drawings
Fig. 1 is the extraction figure of structural granule fat of the present invention and nanometer fat.
Fig. 2 is the 3rd generation nanometer adipose-derived stem cells morphological feature of the invention and Multidirectional Differentiation induction result figure.
In figure, A, B, C are mainly fusiformis for cellular morphology after passage, and minority is triangle or polygon；D, E were the 3rd generation
Cell is through adipogenic induction 14d, it is seen that intracellular to have bright fat drips to be formed, and oil red O dyes visible fat drips and is dyed to cerise；
F, G is after osteogenic induction 21d, it is seen that typical mineralising calcium tubercle is formed, Alizarin red staining Wu's cerise；H, I are into cartilage and lured
Lead after 14d, it is seen that the cell mass of high aggregation sample growth, in patch shape or nodositas, peripheral cell is radial；Tubercle compared with
Greatly, naked eyes are visible；Alcian blue dyeing prompting tubercle and the cell of periphery aggregation are in blueness.
Fig. 3 is the extraction schematic diagram that the present invention is rich in cell factor Fibrin Glue.
Fig. 4 is different time points of the present invention rich in the release cell factor Enzyme-linked Immunosorbent Assay inspection of cell factor Fibrin Glue
Survey result figure.
Fig. 5 is the 3rd generation NFSCs surface antigen flow cytometric analysis results figure of the invention.
Embodiment
Implementation result of the present invention is described in detail with reference to typical case.
Typical case 1
Patient's Wei so-and-so, female, 30 years old, because facial soft tissue depression and profile are uncoordinated medical, it is desirable to which operation improves.In
On January 29th, 2016, row " the Adipose-derived stromal cells cograft material for being rich in cell factor " was local under local anesthesia notes
Penetrate treatment, wherein bilateral temples injection transplantation 45ml, forehead injection transplantation 30ml, bilateral Face and cheek injection transplantation 20ml, chin
Injection transplantation 5ml.12 months meeting contourings of Follow-up After are good, and fuller at soft tissue depression, graft survival is good, face
Skin quality is obviously improved, satisfactory effect.
Typical case 2
Patient what so-and-so, female, 26 years old, because bilateral lower eyelid portion (eyelid buccal groove), decree line (nasolabial groove) soft tissue depression are not assisted
Adjust medical, it is desirable to which operation improves.On March 12nd, 2016, row " was rich in the fat-derived matrix of cell factor under local anesthesia
Cell cograft material " local injection is treated, wherein bilateral eyelid buccal groove injection transplantation 1.6ml, bilateral nasolabial groove injection transplantation
10ml.Follow-up After is shown in that profile is good for 12 months, fuller at soft tissue depression, and graft survival is good, and local skin is smooth, hair
Hole diminishes, satisfactory effect.
Typical case 3
Patient revives so-and-so, female, 25 years old.Because of congenital micromazia and asymmetric examine, it is desirable to which operation improves.In
On November 28th, 2015, row bilateral thigh lipsuction+bilateral breast " was rich in the fat-derived base of cell factor under general anesthesia
" injection transplantation art, wherein left breast inject cell complexes 220ml to cell plastid cograft material, and right breast injection is thin
Born of the same parents' compound 190ml.Follow-up After is shown in that bilateral breast shape is full for 12 months, and graft survival is good, have no graft liquefaction,
The complication such as scleroma, tumour, satisfactory effect.
Claims (2)
1. a kind of preparation of the Adipose-derived stromal cells cograft material rich in cell factor, it is characterised in that preparation side
Method comprises the following steps：
1) acquisition and purifying of lipochondrion group
Outside or lower abdomen in upper lap is chosen, to supply area, to reserve air method lower negative pressure with hand-held 20ml syringes and extract, according to
Patient profiles, select fat for after zone position, local swelling liquid infiltration anesthesia is entered using the porous special fat suction needle of designed, designed
Row suction, is aspirated during suction from deep layer to shallow-layer, and sector is aspirated repeatedly；By the lipochondrion pumped out, using physiological saline
Rinse standby；Take low-speed centrifugal method to obtain and purify lipochondrion group, centrifugal rotational speed 600rpm/min, centrifugation time
2min, operating process whole process follows sterile closed operating principle, and the time of contact of adipose tissue and air is reduced as far as possible；
2) extraction of nanometer adipose stromal cells
A part of lipochondrion is rolled into a ball and smashs nanometer adipose stromal cells of the emulsion process preparation rich in fat stem cell to pieces through machinery, will
Step 1) Grainy fat tissue via hole diameter 0.5mm after preliminary treatment strainer filtering, then by Grainy fat tissue through 2 20mL injection needles
Quickly air exercise 30 times of the fatty special-purpose emulsifying device of cylinder connection nanometer, machinery emulsification is filtered, finally again with the filter screen in 0.5mm apertures
It is 8000rpm/min through rotating speed, it is nanometer that centrifugation time removes the fat emulsification liquid obtained after grease for 5min high speed centrifugations
Fat, the nanometer fat after the emulsification of this part carries more stromal vascular fraction and fat stem cell, is conducive to increasing base
The contact surface of matter vascular components and fat cell, improves the survival rate of fat graft, and machinery emulsification Grainy fat tissue is separated and trained
The mescenchymal stem cell supported and obtained after passing on is referred to as a nanometer adipose-derived stem cells；
3) biological characteristics of nanometer adipose-derived stem cells and Multidirectional Differentiation identification
Through step 2) the nanometer adipose stromal cells after machinery emulsification start after containing more stromal vascular fraction, culture 24h
The cell for having a small amount of fibroblast sample form is dispersed in adherent, and most of non-attached cell is spherical or circular heteroproteose cell, 24-
Liquid is changed after 48h first to remove after non-attached cell, most of attached cell is in roomy flat fibroblast-like cell, with
The extension of incubation time, these attached cells grow in colony shape, and colony is not of uniform size；Cellular morphology is typical shuttle in colony
Shape cell, about 7-10d are up to 80%~90% compacted zone individual layer, and cellular morphology is mainly fusiformis after passage, and the speed of growth is more former
Substantially speeded for cell, if by 1：3 passages, 2-3d can be covered with.Continuous passage culture 10 generation cell does not occur obvious aging
Cell doubling time is averagely about 40h to cell after testing after phenomenon, passage, is respectively obtained for cellular morphology with traditional collagenase digestion
The fat stem cell taken is similar, and the 3rd generation cell is through adipogenic induction 14d, it is seen that intracellular to have bright fat drips to be formed, oil red O dyes
The visible fat drips of color are dyed to cerise；After osteogenic induction 21d, it is seen that typical mineralising calcium tubercle is formed, and Alizarin red staining is in fresh
It is red；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 in put
Penetrate shape；Tubercle is larger, and naked eyes are visible；Alcian blue dyeing prompting tubercle and the cell of periphery aggregation are in blueness；3rd generation nanometer fat
Fat Derived Stem Cells flow cytometry shows that CD29, CD44, CD49d, CD54, CD90 and CD105 are positive, and CD34,
CD45 and CD106 are negative；
4) it is rich in the extraction of cell factor Fibrin Glue
Extract in same individual appropriate venous blood, the sterile negative pressure heparin tube being placed in, using Choukroun ' s method centrifugal rotational speeds
It is that the centrifugation of 12min disposable high-speeds is extracted rich in cell factor Fibrin Glue for 2700rpm/min, centrifugation time；
With 20ml asepsis injectors gather Venous Blood liquid 20-40ml, and closure seal, by sterile centrifuge shield be put into from
In scheming, the 20ml syringes that then will be equipped with Venous Blood liquid are put into sleeve pipe, are centrifuged by 2700rpm/min of rotating speed
12min, is stood and supernatant is abandoned after 3~5min, removes bottom bib, collects intermediate filament protein gel layer, and filtering is removed
Serum, the Fibrin Glue that will be enriched in cell factor collects standby；PRF was carried out within 1~168h periods simultaneously cell because
The detection of sub- release concentration, as a result find VEGF, platelet derived growth factor, transforming growth factor-beta 1,
EGF, interleukin-6, the concentration of Fibroblast collagenase and insulin-like growth factor-i become in rising
Gesture, these cell factors have important effect to promotion organization reparation and regeneration.
2. the clinical practice of the Adipose-derived stromal cells cograft material rich in cell factor as claimed in claim 1,
Characterized in that, pressing certain ratio by nanometer adipose stromal cells, rich in cell factor Fibrin Glue and appropriate Grainy fat tissue
Example is mixed to form composite graft, reparation and correction for clinical upper surface portion, chest and other body surface soft tissue depressions deformity.
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