El aislamiento rápido de BMPR-IB + derivadas de tejido adiposo células estromales de uso en un Defecto La curación Modelo de calota
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
Los principales defectos óseos que resultan de una lesión, infección o cáncer invasivo tienen un impacto significativo en la recuperación y la calidad de vida de un paciente. Existen técnicas para llenar estos defectos con hueso sano de en el propio cuerpo del paciente en otro lugar, pero esta transferencia lleva su propia morbilidad y riesgo de complicaciones 1, 2, 3. Además, algunos defectos son tan grandes o complejas que suficiente hueso de donante no está disponible para llenar el defecto. Los dispositivos protésicos son una opción potencial para el relleno de defectos óseos, pero estos están asociados con varias desventajas, incluyendo el riesgo de infección, un fallo de hardware, y la reacción de cuerpo extraño 4.
Por estas razones, hay un gran interés en la posibilidad de la ingeniería de sustitutos óseos biológicos usando las propias células del paciente 5. Las células del estroma derivadas de tejido adiposo (ASC)tienen potencial para esta aplicación, ya que son abundantemente disponible en el propio tejido graso del paciente y que han demostrado la capacidad de curar defectos óseos mediante la generación de nuevo tejido óseo 6, 7. ASCs son una población diversa de células y varios estudios han demostrado que la selección de marcadores de superficie celular específicos puede producir poblaciones de células con actividad osteogénica mejorada 8, 9. Selección de ASC con el mayor potencial osteogénico aumentaría la probabilidad de que un andamio sembrada con estas células podría regenerar un defecto óseo grande.
Proteína morfogenética de señalización ósea (BMP) es fundamental para la regulación de la diferenciación y formación de hueso 10 y el tipo de BMP Receptor IB (BMPR-IB) se sabe que es importante para la osteogénesis en ASC 11. Recientemente, hemos demostrado que la expresión de BMPR-IB puede be utiliza para seleccionar de ASC con una mayor actividad osteogénica 12. Aquí se demuestra un protocolo para el aislamiento de las ASC derivadas de la grasa humana, seguido de un ensayo de su actividad osteogénica mediante un vivo calota modelo de defecto en BMPR-IB-expresión.
Protocol
Representative Results
Discussion
Los pasos críticos dentro del Protocolo
Durante la cosecha de ASC, el paso crítico es la digestión adecuada de grasa con colagenasa. Digestión inadecuada dará lugar a un bajo rendimiento de ASC. Durante FACS clasificación de células BMPR-IB +, es importante definir cuidadosamente la puerta para la positividad. Definición de puertas demasiado flojo puede dar lugar a poblaciones clasificadas que no son puros. Durante la creación del defecto de la bóveda craneal, es fundamental para perf…
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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