Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery

Lee Lankford, Y. Julia Chen, Zoe Saenz, Priyadarsini Kumar, Connor Long, Diana L Farmer, Aijun Wang

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Background In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). Methods PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). Results PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3–5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. Discussion PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.

Original languageEnglish (US)
Pages (from-to)680-688
Number of pages9
JournalCytotherapy
Volume19
Issue number6
DOIs
StatePublished - Jun 1 2017

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Mesenchymal Stromal Cells
Placenta
Chorionic Villi
Immunophenotyping
Pregnancy
Mycoplasma
Cell Transplantation
Nerve Growth Factors
Cell- and Tissue-Based Therapy
Endotoxins
Plastics
Small Intestine
Extracellular Matrix
Cell Survival
Intercellular Signaling Peptides and Proteins
Flow Cytometry
Swine
Stem Cells
Enzyme-Linked Immunosorbent Assay
Cell Proliferation

Keywords

  • cell delivery
  • cell manufacturing
  • Current Good Manufacturing Practice
  • mesenchymal stromal cells
  • placenta
  • small intestine submucosa

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology
  • Oncology
  • Genetics(clinical)
  • Cell Biology
  • Cancer Research
  • Transplantation

Cite this

Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery. / Lankford, Lee; Chen, Y. Julia; Saenz, Zoe; Kumar, Priyadarsini; Long, Connor; Farmer, Diana L; Wang, Aijun.

In: Cytotherapy, Vol. 19, No. 6, 01.06.2017, p. 680-688.

Research output: Contribution to journalArticle

Lankford, Lee ; Chen, Y. Julia ; Saenz, Zoe ; Kumar, Priyadarsini ; Long, Connor ; Farmer, Diana L ; Wang, Aijun. / Manufacture and preparation of human placenta-derived mesenchymal stromal cells for local tissue delivery. In: Cytotherapy. 2017 ; Vol. 19, No. 6. pp. 680-688.
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abstract = "Background In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). Methods PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). Results PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3–5 × 105 cells/cm2, and seeded cells were >95{\%} viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. Discussion PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.",
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AU - Lankford, Lee

AU - Chen, Y. Julia

AU - Saenz, Zoe

AU - Kumar, Priyadarsini

AU - Long, Connor

AU - Farmer, Diana L

AU - Wang, Aijun

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N2 - Background In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). Methods PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). Results PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3–5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. Discussion PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.

AB - Background In this study we describe the development of a Current Good Manufacturing Practice (CGMP)-compliant process to isolate, expand and bank placenta-derived mesenchymal stromal cells (PMSCs) for use as stem cell therapy. We characterize the viability, proliferation and neuroprotective secretory profile of PMSCs seeded on clinical-grade porcine small intestine submucosa extracellular matrix (SIS-ECM; Cook Biotech). Methods PMSCs were isolated from early gestation placenta chorionic villus tissue via explant culture. Cells were expanded, banked and screened. Purity and expression of markers of pluripotency were determined using flow cytometry. Optimal loading density and viability of PMSCs on SIS-ECM were determined using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) cell proliferation and fluorescent live/dead assays, respectively. Growth factors secretion was analyzed using enzyme-linked immunosorbent assays (ELISA). Results PMSCs were rapidly expanded and banked. Viable Master and Working Cell Banks were stable with minimal decrease in viability at 6 months. All PMSCs were sterile, free from Mycoplasma species, karyotypically normal and had low endotoxin levels. PMSCs were homogeneous by immunophenotyping and expressed little to no pluripotency markers. Optimal loading density on SIS-ECM was 3–5 × 105 cells/cm2, and seeded cells were >95% viable. Neurotrophic factor secretion was detectable from PMSCs seeded on plastic and SIS-ECM with variability between donor lots. Discussion PMSCs from early gestation placental tissues can be rapidly expanded and banked in stable, viable cell banks that are free from contaminating agents, genetically normal and pure. PMSC delivery can be accomplished by using SIS-ECM, which maintains cell viability and protein secretion. Future work in vivo is necessary to optimize cell seeding and transplantation to maximize therapeutic capabilities.

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KW - cell manufacturing

KW - Current Good Manufacturing Practice

KW - mesenchymal stromal cells

KW - placenta

KW - small intestine submucosa

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