BMPR-IB +의 신속한 격리 두개골 결함 치유 모델에서 사용을위한 기질 세포를 지방이는 유래
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
손상, 감염 또는 경부암 인한 주요 골 결손 환자의 회복 및 삶의 질에 큰 영향을 미친다. 다른 기술은 환자의 몸으로부터 뼈 건강 이러한 결함을 채우기 위해 존재하지만,이 이동은 자체 이환율과 합병증 1, 2, 3의 위험도를 전달한다. 또한, 일부 결함이 충분한 기증자 뼈 결함을 채우기 위해 사용할 수없는 너무 크거나 복잡하다. 보철 장치는 뼈 결함을 작성을위한 잠재적 인 옵션이 있지만 이러한 감염 위험, 하드웨어 오류 및 이물 반응 (4) 등 여러 가지 단점과 연결되어 있습니다.
이 때문에, 환자 자신의 세포를 이용한 생물학적 5 골 대체재 공학 가능성에 관심이있다. 지방 유래 기질 세포 (의 ASC)이들은 환자 자신의 지방 조직에서 풍부하게 가능하고 새로운 뼈 조직 (6, 7)를 생성하여 뼈의 결함을 치유 할 수있는 능력을 입증하기 때문에이 애플리케이션에 대한 가능성을 갖는다. 세포의 ASC는 여러 다양한 연구 집단은 특정 세포 표면 마커에 대한 선택하는 향상된 골 형성 활성 (8, 9)와 세포 집단을 생산할 수 있음을 보여 주었다이다. 가장 조골 전위의 ASC를 선택하면 이들 세포 시드 발판 큰 골 결손을 재생할 수있는 가능성을 증가시킬 것이다.
뼈 형태 형성 단백질 (BMP) 시그널링은 골 형성 분화의 ASC (10) 및 (11)에서 골 형성에 중요한 것으로 알려진 BMP 수용체 타입 IB (BMPR-IB)를 조절하는 매우 중요하다. 최근에, 우리는 수 b BMPR-IB의 표현을 보여 주었다전자는 강화 된 골 형성 활동 (12)의 ASC를 위해 선택하는 데 사용됩니다. 여기에서 우리는 생체 내 두개골 결손 모델을 사용하여 골 형성 활동의 분석 뒤에 인간 지방에서의 ASC를 BMPR이-IB가 발현의 분리를위한 프로토콜을 보여줍니다.
Protocol
Representative Results
Discussion
프로토콜 내에서 중요한 단계
의 ASC의 수확하는 동안, 중요한 단계는 콜라겐과 지방의 적절한 소화합니다. 부적절한 소화의 ASC의 낮은 수율 발생합니다. BMPR-IB + 세포를 FACS 정렬 동안 신중 양성하는 게이트를 정의하는 것이 중요하다. 너무 느슨하게 게이트를 정의하는 것은 순수하지 정렬 인구의 원인이 될 수 있습니다. 두개골 결함의 생성 동안, 두개골의 뼈를 통해 결함을 드…
Divulgations
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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