Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect 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

Major bone defects resulting from injury, infection, or invasive cancer have a significant impact on a patient's recovery and quality of life. Techniques exist to fill these defects with healthy bone from elsewhere in the patient's own body, but this transfer carries its own morbidity and risk of complications^1,2,3. Furthermore, some defects are so large or complex that sufficient donor bone is not available to fill the defect. Prosthetic devices are a potential option for filling bony defects but these are associated with several disadvantages including infection ris....

Protocol

NOTE: Samples were obtained from patients who gave informed consent. All protocols were reviewed and approved by the appropriate Stanford University Institutional Review Board. While handling human tissue and cells, always adhere to Biosafety Level 2 (BSL2) precautions, as specified by your institution.

1. Preparation of Reagents

1. Prepare FACS buffer: Add 10 mL FBS, 5 mL Poloxamer 188 and 5 mL Pen-Strep to 500 mL sterile phosphate-buffered saline (PBS).
2. Prepare digest mixture: Add 0.375 g C. hemolyticum collagenase type II powder and 5 mL Poloxamer 188 to 500 mL sterile 199/EBSS medium.
3. Prepare s....

Results

Micro CT scan done on the day of surgery will clearly show the skull defect. At this time there will be no ingrowth into the 4 mm defect. Subsequent scans are obtained over time to quantify the size of the defect over time compared with the baseline. Defects seeded with BMPR-IB+ cells should demonstrate more rapid closure of the defect when compared with BMPR-IB- and Unsorted cells (Figure 5). In addition, the portion of the skull containing the defect can be decalcified .......

Discussion

Critical Steps within the Protocol

During the harvest of ASCs, the critical step is adequate digestion of fat with collagenase. Inadequate digestion will result in a low yield of ASCs. During FACS sorting of BMPR-IB+ cells, it is important to carefully define the gate for positivity. Defining gates too loosely may result in sorted populations that are not pure. During creation of the calvarial defect, it is critical to drill the defect through the bone of the skull but to not advance into the dur.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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