Hurtig Isolering af BMPR-IB + Adipose-afledte stromaceller til brug i en calvariale Defekt Healing Model
Summary
Adipose-derived stromal cells may be useful for engineering new tissue from a patient’s own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.
Abstract
Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient’s own body. The ability to grow bone de novo using a patient’s own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient’s own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.
Introduction
Større knogledefekter skyldes skade, infektion, eller invasiv cancer have en betydelig indvirkning på en patients helbredelse og livskvalitet. Teknikker eksisterer for at udfylde disse fejl med sund knogle fra andre steder i patientens egen krop, men denne overførsel bærer sin egen sygelighed og risiko for komplikationer 1, 2, 3. Desuden er nogle defekter er så stort eller komplekst at tilstrækkelig donor knogle er ikke tilgængelig til at fylde defekten. Proteseindretninger er en potentiel mulighed til fyldning knogledefekter men disse er forbundet med adskillige ulemper, herunder infektionsrisiko, hardwarefejl, og fremmedlegemereaktion 4.
Af disse årsager er der stor interesse i muligheden for engineering biologiske knoglesubstitutter ved hjælp af en patientens egne celler 5. Adipøst afledte stromaceller (ASC'er)har potentiale til denne anvendelse, fordi de er rigeligt til rådighed i patientens eget fedtvæv og de har vist evnen til at helbrede knogledefekter ved at generere nye knoglevæv 6, 7. ASC'er er en forskelligartet population af celler og flere undersøgelser har vist, at selektion for specifikke celleoverflademarkører kan producere cellepopulationer med forøget osteogen aktivitet 8, 9. Valg ASC'er med den højeste osteogene potentiale vil øge sandsynligheden for, at et stillads podet med disse celler kunne regenerere en stor knogledefekt.
Knoglemorfogenetisk protein (BMP) signalering er kritisk for regulering af knogle differentiering og dannelse 10 og BMP receptor type IB (BMPR-IB) vides at være vigtige for osteogenese i ASC'er 11. For nylig har vi vist, at ekspression af BMPR-IB kan be bruges til at vælge for ASC med forbedret osteogen aktivitet 12. Her demonstrerer vi en protokol til isolering af BMPR-IB-udtrykkende ASC'er fra humant fedt efterfulgt af et assay af deres osteogen aktivitet under anvendelse af en in vivo calvariale defekt model.
Protocol
Representative Results
Discussion
Kritiske trin i protokollen
Under høsten af ASC'er, det afgørende skridt er tilstrækkelig fordøjelse af fedt med collagenase. Utilstrækkelig fordøjelse vil resultere i et lavt udbytte af ASC'er. Under FACS sortering af BMPR-IB + -celler, er det vigtigt nøje at definere gate for positivitet. Definition porte for løst kan resultere i sorterede befolkningsgrupper, der ikke er rene. Under oprettelsen af calvariale defekt, er det kritisk at bore defekten gennem knoglen af…
Disclosures
The authors have nothing to disclose.
Acknowledgements
C.D.M. was supported by the American College of Surgeons (ACS) Resident Research Scholarship. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159. M.S.H., H.P.L., and M.T.L. were supported by the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award. H.P.L. was supported by NIH grant R01 GM087609 and a gift from Ingrid Lai and Bill Shu in honor of Anthony Shu. H.P.L. and M.T.L. were supported by the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation. M.T.L. was supported by NIH grants U01 HL099776, R01 DE021683-01, and RC2 DE020771. D.C.W. was supported by NIH grant 1K08DE024269, the Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award.
Materials
| 100 micron cell strainer | Falcon | 352360 | |
| 15 blade scalpel | Miltex | 4-515 | |
| 24 well plate | Corning | 3524 | |
| 40 micron cell strainer | Falcon | 352340 | |
| 50 mL conical centrifuge tubes | Falcon | 352098 | |
| 6-0 Ethilon nylon suture, 18", P-3 needle, | Ethicon | 1698G | |
| Anti-BMPR-IB primary antibody | R&D systems | FAB5051A | |
| BioGel PI surgical gloves | Mölnlycke Health Care | ALA42675Z | |
| Buprenorphine SR | ZooPharm | ||
| Castro-Viejo needle driver | Fine Science Tools | 12565-14 | |
| CD1 nude mouse | Charles River | 086 | |
| Collagenase Type II powder | Gibco | 17101-015 | |
| DMEM medium | Gibco | 10564-011 | |
| Drill: Circular knife 4.0 mm | Xemax Surgical | CK40 | |
| Drill: Z500 Brushless Micromotor | NSK | NSKZ500 | |
| FBS | Gicbo | 10437-077 | |
| Fisherbrand Absorbent Underpads, 20" x 24" | Fisher Scientific | 14-206-62 | |
| Fisherbrand Sterile cotton gauze pad, 4" x 4" | Fisher Scientific | 22-415-469 | |
| Heating pad | Kent Scientific | DCT-20 | |
| Hyclone 199/EBSS medium | GE Life Sciences | SH30253.01 | |
| Isothesia isoflurane | Henry Schein | 050033 | |
| Micro Forceps with teeth | Roboz | RS-5150 | |
| Micro Forceps with teeth | Roboz | RS-5150 | |
| Paraffin film (Parafilm) | Bemis | PM996 | |
| PBS | Gibco | 10010-023 | |
| Pen-Strep | Gibco | 15140-122 | |
| PLGA scaffolds | Proprietary Formulation | ||
| Poloxamer 188, 10% | Sigma | P5556-100ML | |
| Polylined Sterile Field, 18" x 24" | Busse Hospital Disposables | 696 | Cut a rectangular hole of the appropriate size |
| Polysucrose Solution: Histopaque 1119 | Sigma | 11191 | |
| Povidone Iodine Prep Solution | Medline | MDS093944H | |
| Puralube petrolatum ophthalmic ointment, 1/8 oz. tube | Dechra Veterinary Products | ||
| RBC lysis buffer | Sigma | 11814389001 | |
| Webcol alcohol prep swabs | Covidien | 6818 |
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