Comparison of human bone marrow mononuclear cell isolation methods for creating tissue-engineered vascular grafts: Novel filter system versus traditional density centrifugation method

Narutoshi Hibino, Ani Nalbandian, Lesley Devine, Rajendra Sawh Martinez, Edward McGillicuddy, Tai Yi, Safa Karandish, Girolamo A. Ortolano, Toshiharu Shin'Oka, Edward Snyder, Christopher K. Breuer

Research output: Contribution to journalArticle

Abstract

Introduction: We created the first tissue-engineered vascular graft (TEVG) to be successfully used in humans. The TEVG is made by seeding autologous bone marrow-derived mononuclear cells (BM-MNCs) onto a biodegradable tubular scaffold fabricated from polyglycolic-acid mesh coated with a 50:50 copolymer of poly-L-lactide and-ε-caprolactone. In the initial clinical study, the BM-MNCs were isolated using a Ficoll density centrifugation method. Use of this cell isolation technique is problematic in that it is performed using an open system and therefore is susceptible to contamination. As a first step toward creating a closed system for assembling a TEVG, we evaluated the use of a filter-based method for isolating BM-MNCs and compared it to density centrifugation in Ficoll. Methods: BM-MNCs were isolated from human BM using density centrifugation in Ficoll or a filter-based method. BM-MNCs were seeded onto biodegradable tubular scaffold and incubated for 24h before implantation. The TEVG were implanted as inferior vena cava interposition grafts in SCID/bg mice (n=24) using microsurgical technique. Grafts were followed with ultrasonography and computed tomography-angiography. Ten weeks after implantation the TEVG were explanted and examined using histology and immunohistochemistry. Results: Both methods isolated similar number of cells (Ficoll: 8.5±6.6×106/mL, Filter: 6.6±3. 5×106/mL; p=0.686) with similar viability as assayed using fluorescence-activated cell sorting (FACS) (Ficoll: 97.0%±1.5%, Filter: 95.9%±3.0%; p=0.339). FACS analysis demonstrated that the fraction of lymphocytes and monocytes to total cells was lower in the filter group (CD4 in Ficoll: 8.9%±1.1%, CD4 in Filter: 3.5%±0.8%; p=0.002, CD8 in Ficoll: 9.4%±2.1%, CD8 in Filter: 3.9%±1.4%; p=0.021, Monocyte in Ficoll: 6.9%±1.0%, Monocyte in Filter: 2.7%±1.0%; p=0.008), consistent with granulocyte contamination (Ficoll: 46.6±2.7×10 6/mL, Filter: 58.1±5.2×106/mL; p<0.001). The ratio of stem cells to BM-MNCs was comparable between groups. There were no statistically significant differences with regard to TEVG patency and morphology between groups. Both methods of cell isolation produced neovessels with similar histology. Conclusion: Filter-based BM-MNC isolation is comparable to BM-MNC isolation using density centrifugation in Ficoll for TEVG assembly. The filter-based cell isolation technique has the added advantage of the potential to create a closed disposable system.

Original languageEnglish (US)
Pages (from-to)993-998
Number of pages6
JournalTissue Engineering - Part C: Methods
Volume17
Issue number10
DOIs
StatePublished - Oct 1 2011
Externally publishedYes

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Blood Vessel Prosthesis
Ficoll
Centrifugation
Cell Separation
Grafts
Bone Marrow Cells
Bone
Tissue
Bone Marrow
Histology
Monocytes
Sorting
Scaffolds
Contamination
Fluorescence
Flow Cytometry
Vascular Patency
Ultrasonography
Polyglycolic Acid
Transplants

ASJC Scopus subject areas

  • Biomedical Engineering
  • Bioengineering
  • Medicine (miscellaneous)
  • Medicine(all)

Cite this

Comparison of human bone marrow mononuclear cell isolation methods for creating tissue-engineered vascular grafts : Novel filter system versus traditional density centrifugation method. / Hibino, Narutoshi; Nalbandian, Ani; Devine, Lesley; Martinez, Rajendra Sawh; McGillicuddy, Edward; Yi, Tai; Karandish, Safa; Ortolano, Girolamo A.; Shin'Oka, Toshiharu; Snyder, Edward; Breuer, Christopher K.

In: Tissue Engineering - Part C: Methods, Vol. 17, No. 10, 01.10.2011, p. 993-998.

Research output: Contribution to journalArticle

Hibino, N, Nalbandian, A, Devine, L, Martinez, RS, McGillicuddy, E, Yi, T, Karandish, S, Ortolano, GA, Shin'Oka, T, Snyder, E & Breuer, CK 2011, 'Comparison of human bone marrow mononuclear cell isolation methods for creating tissue-engineered vascular grafts: Novel filter system versus traditional density centrifugation method', Tissue Engineering - Part C: Methods, vol. 17, no. 10, pp. 993-998. https://doi.org/10.1089/ten.tec.2011.0110
Hibino, Narutoshi ; Nalbandian, Ani ; Devine, Lesley ; Martinez, Rajendra Sawh ; McGillicuddy, Edward ; Yi, Tai ; Karandish, Safa ; Ortolano, Girolamo A. ; Shin'Oka, Toshiharu ; Snyder, Edward ; Breuer, Christopher K. / Comparison of human bone marrow mononuclear cell isolation methods for creating tissue-engineered vascular grafts : Novel filter system versus traditional density centrifugation method. In: Tissue Engineering - Part C: Methods. 2011 ; Vol. 17, No. 10. pp. 993-998.
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T1 - Comparison of human bone marrow mononuclear cell isolation methods for creating tissue-engineered vascular grafts

T2 - Novel filter system versus traditional density centrifugation method

AU - Hibino, Narutoshi

AU - Nalbandian, Ani

AU - Devine, Lesley

AU - Martinez, Rajendra Sawh

AU - McGillicuddy, Edward

AU - Yi, Tai

AU - Karandish, Safa

AU - Ortolano, Girolamo A.

AU - Shin'Oka, Toshiharu

AU - Snyder, Edward

AU - Breuer, Christopher K.

PY - 2011/10/1

Y1 - 2011/10/1

N2 - Introduction: We created the first tissue-engineered vascular graft (TEVG) to be successfully used in humans. The TEVG is made by seeding autologous bone marrow-derived mononuclear cells (BM-MNCs) onto a biodegradable tubular scaffold fabricated from polyglycolic-acid mesh coated with a 50:50 copolymer of poly-L-lactide and-ε-caprolactone. In the initial clinical study, the BM-MNCs were isolated using a Ficoll density centrifugation method. Use of this cell isolation technique is problematic in that it is performed using an open system and therefore is susceptible to contamination. As a first step toward creating a closed system for assembling a TEVG, we evaluated the use of a filter-based method for isolating BM-MNCs and compared it to density centrifugation in Ficoll. Methods: BM-MNCs were isolated from human BM using density centrifugation in Ficoll or a filter-based method. BM-MNCs were seeded onto biodegradable tubular scaffold and incubated for 24h before implantation. The TEVG were implanted as inferior vena cava interposition grafts in SCID/bg mice (n=24) using microsurgical technique. Grafts were followed with ultrasonography and computed tomography-angiography. Ten weeks after implantation the TEVG were explanted and examined using histology and immunohistochemistry. Results: Both methods isolated similar number of cells (Ficoll: 8.5±6.6×106/mL, Filter: 6.6±3. 5×106/mL; p=0.686) with similar viability as assayed using fluorescence-activated cell sorting (FACS) (Ficoll: 97.0%±1.5%, Filter: 95.9%±3.0%; p=0.339). FACS analysis demonstrated that the fraction of lymphocytes and monocytes to total cells was lower in the filter group (CD4 in Ficoll: 8.9%±1.1%, CD4 in Filter: 3.5%±0.8%; p=0.002, CD8 in Ficoll: 9.4%±2.1%, CD8 in Filter: 3.9%±1.4%; p=0.021, Monocyte in Ficoll: 6.9%±1.0%, Monocyte in Filter: 2.7%±1.0%; p=0.008), consistent with granulocyte contamination (Ficoll: 46.6±2.7×10 6/mL, Filter: 58.1±5.2×106/mL; p<0.001). The ratio of stem cells to BM-MNCs was comparable between groups. There were no statistically significant differences with regard to TEVG patency and morphology between groups. Both methods of cell isolation produced neovessels with similar histology. Conclusion: Filter-based BM-MNC isolation is comparable to BM-MNC isolation using density centrifugation in Ficoll for TEVG assembly. The filter-based cell isolation technique has the added advantage of the potential to create a closed disposable system.

AB - Introduction: We created the first tissue-engineered vascular graft (TEVG) to be successfully used in humans. The TEVG is made by seeding autologous bone marrow-derived mononuclear cells (BM-MNCs) onto a biodegradable tubular scaffold fabricated from polyglycolic-acid mesh coated with a 50:50 copolymer of poly-L-lactide and-ε-caprolactone. In the initial clinical study, the BM-MNCs were isolated using a Ficoll density centrifugation method. Use of this cell isolation technique is problematic in that it is performed using an open system and therefore is susceptible to contamination. As a first step toward creating a closed system for assembling a TEVG, we evaluated the use of a filter-based method for isolating BM-MNCs and compared it to density centrifugation in Ficoll. Methods: BM-MNCs were isolated from human BM using density centrifugation in Ficoll or a filter-based method. BM-MNCs were seeded onto biodegradable tubular scaffold and incubated for 24h before implantation. The TEVG were implanted as inferior vena cava interposition grafts in SCID/bg mice (n=24) using microsurgical technique. Grafts were followed with ultrasonography and computed tomography-angiography. Ten weeks after implantation the TEVG were explanted and examined using histology and immunohistochemistry. Results: Both methods isolated similar number of cells (Ficoll: 8.5±6.6×106/mL, Filter: 6.6±3. 5×106/mL; p=0.686) with similar viability as assayed using fluorescence-activated cell sorting (FACS) (Ficoll: 97.0%±1.5%, Filter: 95.9%±3.0%; p=0.339). FACS analysis demonstrated that the fraction of lymphocytes and monocytes to total cells was lower in the filter group (CD4 in Ficoll: 8.9%±1.1%, CD4 in Filter: 3.5%±0.8%; p=0.002, CD8 in Ficoll: 9.4%±2.1%, CD8 in Filter: 3.9%±1.4%; p=0.021, Monocyte in Ficoll: 6.9%±1.0%, Monocyte in Filter: 2.7%±1.0%; p=0.008), consistent with granulocyte contamination (Ficoll: 46.6±2.7×10 6/mL, Filter: 58.1±5.2×106/mL; p<0.001). The ratio of stem cells to BM-MNCs was comparable between groups. There were no statistically significant differences with regard to TEVG patency and morphology between groups. Both methods of cell isolation produced neovessels with similar histology. Conclusion: Filter-based BM-MNC isolation is comparable to BM-MNC isolation using density centrifugation in Ficoll for TEVG assembly. The filter-based cell isolation technique has the added advantage of the potential to create a closed disposable system.

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