ORIGINAL ARTICLE
Comparison of the Matrix Synthesizing Abilities of Human Adipose-Derived Stromal Vascular Fraction Cells and Fibroblasts Soo-Hye Shin, MD, MSc, Tae Kyoung Yun, MD, Seung-Kyu Han, MD, PhD, Seong-Ho Jeong, MD, PhD, Eun-Sang Dhong, MD, PhD, and Woo-Kyung Kim, MD, PhD Abstract: For facial soft tissue augmentation or wound coverage using tissue-engineering technology, cultured fibroblasts have been most commonly used as key cells and their properties have been extensively studied. Clinical strategies based on human cultured fibroblasts, however, require Food and Drug Administration (FDA) approval for the facilities and the procedures used and time-consuming culture. Adipose tissue-derived stromal vascular fraction (SVF) cells may be a reliable alternative to fibroblasts because they are easily harvested by liposuction and do not require culture or FDA approval. No quantitative standard governing their use has, however, been issued. The purpose of this study was to quantitatively compare matrix-forming abilities of SVF cells and fibroblasts. Human dermal fibroblasts were obtained from the dermis of 10 healthy adults and cultured, and SVF cells were obtained from 10 patients who underwent liposuction. Monolayer and suspension cell cultures were performed using both cell types for 3 days. Cell proliferations, collagen synthesis levels, and glycosaminoglycan levels were compared using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay, a collagen type I carboxyterminal propeptide enzyme immunoassay, and the Blyscan Dye method, respectively. Cell proliferation ratios (fibroblasts versus SVF cells) in monolayer and suspension cultures were 1:0.75 and 1:0.99, respectively; collagen synthesis ratios in monolayer and suspension cultures were 1:0.50 and 1:0.52, respectively; and glycosaminoglycan production ratios were 1:0.70 and 1:0.74, respectively. The results of this in vitro study indicate that SVF cells have 50–74% of the matrix-forming ability of fibroblasts. Key Words: Fibroblast, stromal vascular fraction cell, tissue engineering (J Craniofac Surg 2015;26: 1246–1250)
S
oft tissue defects are a common and often devastating problem, especially in patients involving the face. Attempts to restore soft tissue defects include the grafting of autologous, allogenic, and/or From the Department of Plastic Surgery, Korea University College of Medicine, Seoul, Korea. Received November 24, 2014. Accepted for publication January 30, 2015. Address correspondence and reprint requests to Seung-Kyu Han, MD, PhD, Department of Plastic Surgery, Korea University Guro Hospital, 148 Guro-Dong, Guro-Ku, Seoul 152-703, Korea. E-mail: [email protected] The authors report no conflicts of interest. Copyright # 2015 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000001828
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artificial tissues.1,2 Recently, tissue-engineering technologies based on combinations of cells and scaffolds have also been clinically applied. These technologies are straightforward, relatively noninvasive, reduce operation times, and minimize donor site morbidities.3 The use of cultured fibroblasts is representative of the tissue engineering of soft tissue, and numerous previous studies have shown that fibroblasts are suitable for the treatment of chronic wounds and other soft tissue defects.4,5 In addition, there are a wide variety of commercial tissue-engineering products based on fibroblast use. If cultured fibroblasts are, however, to be used for clinical purposes, Food and Drug Administration (FDA)-approved facilities and techniques and lengthy culture periods are required, and these limitations make it difficult to consider using cultured fibroblasts for clinical purposes.2,6 Over the past 4 decades, the perspective of the medical community regarding the use of adipose tissue has changed dramatically.7,8 In 2013, Lee et al showed that the transplantation of human adipose-derived stromal vascular fraction (SVF) cells results in the formation of well-vascularized fibrous connective tissue. Furthermore, they suggested that SVF cells should be considered for the replacement of dermal fibroblasts because these cells can be obtained easily in large volume from adipose tissue by liposuction without cell culture.2,9 Although SVF cells could be considered a reliable source for the tissue engineering of soft tissue, unlike fibroblasts, no quantitative standard has been issued regarding the amounts required for effective soft tissue restoration. To use SVF cells effectively in the clinical setting, the tissue-forming ability of SVF cells must be evaluated. In the case of fibroblasts, several commercial products are available and standard guidelines with respect to cell numbers have been issued. In contrast, SVF cells are heterogeneous in nature and include endothelial cells, fibroblasts, various precursor cells, and stem cells. This multicellular character of SVF cells makes it difficult to predict soft tissue formation results after transplantation. At the time of this writing, the amounts of SVF cells used therapeutically depend on the empirical experiences of individual physicians, and no previous study has compared the soft tissueforming abilities of SVF cells and fibroblasts. The purpose of this in vitro study was to compare the abilities of fibroblasts and SVF cells to produce extracellular matrices (ECMs). In particular, cell proliferations, collagen synthesis, and glycosaminoglycan (GAG) production levels of these 2 cell types were compared. We hoped that the results of this study would give physicians using SVF cell-based therapy helpful information regarding the amounts of SVF cells required for transplantation.
METHODS This study was approved by the Institutional Review Board of Korea University Guro Hospital (approval number: KUGH13214).
The Journal of Craniofacial Surgery
Volume 26, Number 4, June 2015
Copyright © 2015 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery
Volume 26, Number 4, June 2015
SVF Cells and Fibroblasts
Culture of Dermal Fibroblasts Dermal fibroblasts were obtained from cryopreserved cells derived from the dermis of 10 healthy adults who had provided informed consent for their cells to be used for research purposes. Fibroblasts were spread evenly over the surface of a 100-mm cell culture plate (Corning, New York). Twelve milliliters of Dulbecco’s modified Eagle medium/Ham’s F-12 nutrient (DMEM/F-12; Gibco; Grand Island, NY) containing 10% fetal bovine serum (FBS; Gibco) and 25 mg/mL of gentamycin was added. Cells were then grown to confluence. All cultured cells were grown in a 5% CO2/95% air atmosphere at 100% humidity and 378C. Fibroblasts were dissociated by trypsinization, diluted 2.7-fold in Mg2þ and Ca2þ-free Dulbecco’s phosphate-buffered saline (DPBS; Gibco), and collected by centrifugation at 450 g for 17 min. They were then washed twice in 40 mL of DPBS. Cell densities were determined using a hemocytometer and cell viabilities were assessed using a trypan blue dye exclusion assay. Two-passage cells were used in all the experiments.
Isolation of Human Adipose-Derived Stromal Vascular Fraction Cells Abdominal adipose tissues were obtained from 10 patients who underwent liposuction using a 3-mm inner diameter cannula. The obtained samples were rinsed with DPBS, incubated in DMEM/F12 containing 0.075% type I collagenase for 2 h at 378C, and centrifuged at 300 g for 10 min at 48C. The top lipid layer was removed and the pellets obtained were treated with 160 mM NH4Cl for 10 min to lyse red blood cells. The remaining cells were washed twice in 40 mL DPBS, resuspended in 5 mL DPBS, and filtered through a 100 mm nylon mesh. Cell densities were determined using a hemocytometer and cell viabilities were assessed using a trypan blue dye exclusion assay.
Monolayer Culture Study For cell proliferation assays, cultured fibroblasts or isolated SVF cells were seeded evenly onto 96-well culture plates (96-well surface-treated cell culture plate; SPL Life Sciences, Pocheon, Korea) at 1.5 103 cells/well in DMEM/F-12 containing 5% FBS. For collagen synthesis and GAG production assays, cells were seeded evenly onto 24-well culture plates (24-well surfacetreated cell culture plates; SPL Life Sciences) at 1.0 104 cells/well in DMEM/F-12 containing 5% FBS. Cells were then incubated at 378C in a 100% humidified 5% CO2 atmosphere for 3 days.
Cell Proliferation
FIGURE 1. The proliferations of fibroblasts and SVF cells in monolayer and suspension cultures (P < 0.05). SVF, stromal vascular fraction.
phosphatase conjugates for 50 min. The reaction was then stopped and collagen synthesis was determined by measuring absorbance at 405 nm.
Glycosaminoglycan Synthesis Levels of sulfated GAG were measured in culture media using Blyscan Dye ReagentTM (Biocolor Ltd.; Newtownabbey, UK) according to the manufacturer’s instructions. Briefly, 100 mL aliquots of diluted culture supernatants and 1 mL of dye reagent were added to 1.5 mL microcentrifuge tubes and mixed for 30 min. When the insoluble GAG–dye complex formed, it was separated from the remaining excess soluble unbound dye by centrifuging tubes at 1000 g for 10 min. Supernatants were then discarded and 1 mL of Blyscan Dissociation Reagent (Biocolor Ltd.) was added to each tube. GAG levels were determined by measured absorbance at 656 nm using a spectrophotometer.
Suspension Culture Study For cell proliferation assays, cultured fibroblasts or isolated SVF cells were seeded evenly onto 96-well culture plates (96-well nontreated cell culture plates; SPL Life Sciences), and for the
Cell proliferation was determined using a 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, 10 mL of MTT (5 mg/mL) was added to 100 mL of a cell monolayer in a 96-well plate and incubated for 3 h at 378C. Next, 100 mL of 0.04 M HCl in propan-2-ol was added to each well and mixed thoroughly to dissolve the blue formazan crystals. Absorbance was measured using a test wavelength of 570 mm and a reference wavelength of 630 mm using an enzyme-linked immunosorbent assay reader. Samples were analyzed in triplicate and results were averaged.
Collagen Synthesis To measure collagen production, a collagen type I carboxyterminal propeptide enzyme (CICP) immunoassay was performed using the Metra CICP kit (Quidel, San Diego, CA). Briefly, 100 mL of diluted culture supernatants was added to each well of monoclonal anti-CICP antibody-coated plates, and incubated at room temperature for 2 h. Rabbit anti-CICP antiserum (100 mL) was then added for 50 min followed by 100 mL goat anti-rabbit alkaline #
2015 Mutaz B. Habal, MD
FIGURE 2. Proliferation ratios of fibroblasts and SVF cells. SVF, stromal vascular fraction.
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Copyright © 2015 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery
Shin et al
Volume 26, Number 4, June 2015
collagen synthesis and GAG production assays, cells were seeded evenly onto 24-well culture plates (24-well nontreated cell culture plates; SPL Life Sciences). All conditions, including culture medium, culture conditions, and viability determinations, were identical to those used for monolayer culture. Cell proliferation, collagen synthesis, and GAG production were determined using the methods described above.
Statistical Analyses Results are expressed as means standard deviations. Statistical comparisons were performed using the Mann–Whitney U-test, and P-values of
Comparison of the Matrix Synthesizing Abilities of Human Adipose-Derived Stromal Vascular Fraction Cells and Fibroblasts Soo-Hye Shin, MD, MSc, Tae Kyoung Yun, MD, Seung-Kyu Han, MD, PhD, Seong-Ho Jeong, MD, PhD, Eun-Sang Dhong, MD, PhD, and Woo-Kyung Kim, MD, PhD Abstract: For facial soft tissue augmentation or wound coverage using tissue-engineering technology, cultured fibroblasts have been most commonly used as key cells and their properties have been extensively studied. Clinical strategies based on human cultured fibroblasts, however, require Food and Drug Administration (FDA) approval for the facilities and the procedures used and time-consuming culture. Adipose tissue-derived stromal vascular fraction (SVF) cells may be a reliable alternative to fibroblasts because they are easily harvested by liposuction and do not require culture or FDA approval. No quantitative standard governing their use has, however, been issued. The purpose of this study was to quantitatively compare matrix-forming abilities of SVF cells and fibroblasts. Human dermal fibroblasts were obtained from the dermis of 10 healthy adults and cultured, and SVF cells were obtained from 10 patients who underwent liposuction. Monolayer and suspension cell cultures were performed using both cell types for 3 days. Cell proliferations, collagen synthesis levels, and glycosaminoglycan levels were compared using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay, a collagen type I carboxyterminal propeptide enzyme immunoassay, and the Blyscan Dye method, respectively. Cell proliferation ratios (fibroblasts versus SVF cells) in monolayer and suspension cultures were 1:0.75 and 1:0.99, respectively; collagen synthesis ratios in monolayer and suspension cultures were 1:0.50 and 1:0.52, respectively; and glycosaminoglycan production ratios were 1:0.70 and 1:0.74, respectively. The results of this in vitro study indicate that SVF cells have 50–74% of the matrix-forming ability of fibroblasts. Key Words: Fibroblast, stromal vascular fraction cell, tissue engineering (J Craniofac Surg 2015;26: 1246–1250)
S
oft tissue defects are a common and often devastating problem, especially in patients involving the face. Attempts to restore soft tissue defects include the grafting of autologous, allogenic, and/or From the Department of Plastic Surgery, Korea University College of Medicine, Seoul, Korea. Received November 24, 2014. Accepted for publication January 30, 2015. Address correspondence and reprint requests to Seung-Kyu Han, MD, PhD, Department of Plastic Surgery, Korea University Guro Hospital, 148 Guro-Dong, Guro-Ku, Seoul 152-703, Korea. E-mail: [email protected] The authors report no conflicts of interest. Copyright # 2015 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000001828
1246
artificial tissues.1,2 Recently, tissue-engineering technologies based on combinations of cells and scaffolds have also been clinically applied. These technologies are straightforward, relatively noninvasive, reduce operation times, and minimize donor site morbidities.3 The use of cultured fibroblasts is representative of the tissue engineering of soft tissue, and numerous previous studies have shown that fibroblasts are suitable for the treatment of chronic wounds and other soft tissue defects.4,5 In addition, there are a wide variety of commercial tissue-engineering products based on fibroblast use. If cultured fibroblasts are, however, to be used for clinical purposes, Food and Drug Administration (FDA)-approved facilities and techniques and lengthy culture periods are required, and these limitations make it difficult to consider using cultured fibroblasts for clinical purposes.2,6 Over the past 4 decades, the perspective of the medical community regarding the use of adipose tissue has changed dramatically.7,8 In 2013, Lee et al showed that the transplantation of human adipose-derived stromal vascular fraction (SVF) cells results in the formation of well-vascularized fibrous connective tissue. Furthermore, they suggested that SVF cells should be considered for the replacement of dermal fibroblasts because these cells can be obtained easily in large volume from adipose tissue by liposuction without cell culture.2,9 Although SVF cells could be considered a reliable source for the tissue engineering of soft tissue, unlike fibroblasts, no quantitative standard has been issued regarding the amounts required for effective soft tissue restoration. To use SVF cells effectively in the clinical setting, the tissue-forming ability of SVF cells must be evaluated. In the case of fibroblasts, several commercial products are available and standard guidelines with respect to cell numbers have been issued. In contrast, SVF cells are heterogeneous in nature and include endothelial cells, fibroblasts, various precursor cells, and stem cells. This multicellular character of SVF cells makes it difficult to predict soft tissue formation results after transplantation. At the time of this writing, the amounts of SVF cells used therapeutically depend on the empirical experiences of individual physicians, and no previous study has compared the soft tissueforming abilities of SVF cells and fibroblasts. The purpose of this in vitro study was to compare the abilities of fibroblasts and SVF cells to produce extracellular matrices (ECMs). In particular, cell proliferations, collagen synthesis, and glycosaminoglycan (GAG) production levels of these 2 cell types were compared. We hoped that the results of this study would give physicians using SVF cell-based therapy helpful information regarding the amounts of SVF cells required for transplantation.
METHODS This study was approved by the Institutional Review Board of Korea University Guro Hospital (approval number: KUGH13214).
The Journal of Craniofacial Surgery
Volume 26, Number 4, June 2015
Copyright © 2015 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery
Volume 26, Number 4, June 2015
SVF Cells and Fibroblasts
Culture of Dermal Fibroblasts Dermal fibroblasts were obtained from cryopreserved cells derived from the dermis of 10 healthy adults who had provided informed consent for their cells to be used for research purposes. Fibroblasts were spread evenly over the surface of a 100-mm cell culture plate (Corning, New York). Twelve milliliters of Dulbecco’s modified Eagle medium/Ham’s F-12 nutrient (DMEM/F-12; Gibco; Grand Island, NY) containing 10% fetal bovine serum (FBS; Gibco) and 25 mg/mL of gentamycin was added. Cells were then grown to confluence. All cultured cells were grown in a 5% CO2/95% air atmosphere at 100% humidity and 378C. Fibroblasts were dissociated by trypsinization, diluted 2.7-fold in Mg2þ and Ca2þ-free Dulbecco’s phosphate-buffered saline (DPBS; Gibco), and collected by centrifugation at 450 g for 17 min. They were then washed twice in 40 mL of DPBS. Cell densities were determined using a hemocytometer and cell viabilities were assessed using a trypan blue dye exclusion assay. Two-passage cells were used in all the experiments.
Isolation of Human Adipose-Derived Stromal Vascular Fraction Cells Abdominal adipose tissues were obtained from 10 patients who underwent liposuction using a 3-mm inner diameter cannula. The obtained samples were rinsed with DPBS, incubated in DMEM/F12 containing 0.075% type I collagenase for 2 h at 378C, and centrifuged at 300 g for 10 min at 48C. The top lipid layer was removed and the pellets obtained were treated with 160 mM NH4Cl for 10 min to lyse red blood cells. The remaining cells were washed twice in 40 mL DPBS, resuspended in 5 mL DPBS, and filtered through a 100 mm nylon mesh. Cell densities were determined using a hemocytometer and cell viabilities were assessed using a trypan blue dye exclusion assay.
Monolayer Culture Study For cell proliferation assays, cultured fibroblasts or isolated SVF cells were seeded evenly onto 96-well culture plates (96-well surface-treated cell culture plate; SPL Life Sciences, Pocheon, Korea) at 1.5 103 cells/well in DMEM/F-12 containing 5% FBS. For collagen synthesis and GAG production assays, cells were seeded evenly onto 24-well culture plates (24-well surfacetreated cell culture plates; SPL Life Sciences) at 1.0 104 cells/well in DMEM/F-12 containing 5% FBS. Cells were then incubated at 378C in a 100% humidified 5% CO2 atmosphere for 3 days.
Cell Proliferation
FIGURE 1. The proliferations of fibroblasts and SVF cells in monolayer and suspension cultures (P < 0.05). SVF, stromal vascular fraction.
phosphatase conjugates for 50 min. The reaction was then stopped and collagen synthesis was determined by measuring absorbance at 405 nm.
Glycosaminoglycan Synthesis Levels of sulfated GAG were measured in culture media using Blyscan Dye ReagentTM (Biocolor Ltd.; Newtownabbey, UK) according to the manufacturer’s instructions. Briefly, 100 mL aliquots of diluted culture supernatants and 1 mL of dye reagent were added to 1.5 mL microcentrifuge tubes and mixed for 30 min. When the insoluble GAG–dye complex formed, it was separated from the remaining excess soluble unbound dye by centrifuging tubes at 1000 g for 10 min. Supernatants were then discarded and 1 mL of Blyscan Dissociation Reagent (Biocolor Ltd.) was added to each tube. GAG levels were determined by measured absorbance at 656 nm using a spectrophotometer.
Suspension Culture Study For cell proliferation assays, cultured fibroblasts or isolated SVF cells were seeded evenly onto 96-well culture plates (96-well nontreated cell culture plates; SPL Life Sciences), and for the
Cell proliferation was determined using a 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, 10 mL of MTT (5 mg/mL) was added to 100 mL of a cell monolayer in a 96-well plate and incubated for 3 h at 378C. Next, 100 mL of 0.04 M HCl in propan-2-ol was added to each well and mixed thoroughly to dissolve the blue formazan crystals. Absorbance was measured using a test wavelength of 570 mm and a reference wavelength of 630 mm using an enzyme-linked immunosorbent assay reader. Samples were analyzed in triplicate and results were averaged.
Collagen Synthesis To measure collagen production, a collagen type I carboxyterminal propeptide enzyme (CICP) immunoassay was performed using the Metra CICP kit (Quidel, San Diego, CA). Briefly, 100 mL of diluted culture supernatants was added to each well of monoclonal anti-CICP antibody-coated plates, and incubated at room temperature for 2 h. Rabbit anti-CICP antiserum (100 mL) was then added for 50 min followed by 100 mL goat anti-rabbit alkaline #
2015 Mutaz B. Habal, MD
FIGURE 2. Proliferation ratios of fibroblasts and SVF cells. SVF, stromal vascular fraction.
1247
Copyright © 2015 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery
Shin et al
Volume 26, Number 4, June 2015
collagen synthesis and GAG production assays, cells were seeded evenly onto 24-well culture plates (24-well nontreated cell culture plates; SPL Life Sciences). All conditions, including culture medium, culture conditions, and viability determinations, were identical to those used for monolayer culture. Cell proliferation, collagen synthesis, and GAG production were determined using the methods described above.
Statistical Analyses Results are expressed as means standard deviations. Statistical comparisons were performed using the Mann–Whitney U-test, and P-values of
