Development of novel perfusion chamber to retain nonadherent cells and its use for comparison of human "mobilized" peripheral blood mononuclear cell cultures with and without irradiated bone marrow stroma

Craig E. Sandstrom, James G. Bender, William M Miller, E. Terry Papoutsakis*

*Corresponding author for this work

Research output: Contribution to journalReview article

Abstract

Perfusion and static cultures of peripheral blood (PB) mononuclear cells (MNCs), obtained from patients following stem cell mobilization, were supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF) and compared with and without a preformed irradiated allogeneic bone marrow stromal layer. Perfusion cultures without a stromal layer effectively retained nonadherent cells through the use of a novel "grooved" perfusion chamber, which was designed with minimal mass transfer barriers in order to achieve a well-defined culture environment. The grooved chamber allowed easy and efficient culture inoculation and cell recovery. Average maximum expansion of CFU-GM (colony-forming unit granulocytemacrophage) cells was observed on day 10 for all cultures. Perfusion cultures had a maximum CFU-GM expansion of 17- and 19-fold with and without a stromal layer, respectively. In contrast, static cultures had a maximum CFU-GM expansion of 18- and 13-fold with and without a stromal layer, respectively. Average long-term-culture initiating cell (LTC-IC) numbers on day 15 were 34% and 64% of input in stroma-containing and stromafree perfusion cultures and 12% and 11% of input in stroma-containing and stroma-free static cultures, respectively. Thus, perfusion enhanced CFU-GM expansion and LTC-IC maintenance more for the stroma-free cultures than for stroma-containing cultures. This was surprising because analysis of medium supernatants indicated that the stroma-containing cultures were metabolically more active than the stroma-free cultures. In view of their equivalent, if not superior, performance compared to stroma-containing cultures, stroma-free perfusion cultures may offer significant advantages for potential clinical applications.

Original languageEnglish (US)
Pages (from-to)493-504
Number of pages12
JournalBiotechnology and Bioengineering
Volume50
Issue number5
StatePublished - Jun 5 1996

Fingerprint

Cell culture
Blood Cells
Bone
Blood
Cell Culture Techniques
Perfusion
Bone Marrow
Stem cells
Stem Cells
Stem Cell Factor
Interleukin-3
Granulocyte Colony-Stimulating Factor
Interleukin-6
Hematopoietic Stem Cell Mobilization
Mass transfer
Recovery
Cell Count
Maintenance

Keywords

  • Bone marrow stroma
  • Cell retention
  • Hematopoietic cultures
  • Mononuclear cell cultures
  • Perfusion chamber

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

@article{2761390fd0f646639bfb4c2693024244,
title = "Development of novel perfusion chamber to retain nonadherent cells and its use for comparison of human {"}mobilized{"} peripheral blood mononuclear cell cultures with and without irradiated bone marrow stroma",
abstract = "Perfusion and static cultures of peripheral blood (PB) mononuclear cells (MNCs), obtained from patients following stem cell mobilization, were supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF) and compared with and without a preformed irradiated allogeneic bone marrow stromal layer. Perfusion cultures without a stromal layer effectively retained nonadherent cells through the use of a novel {"}grooved{"} perfusion chamber, which was designed with minimal mass transfer barriers in order to achieve a well-defined culture environment. The grooved chamber allowed easy and efficient culture inoculation and cell recovery. Average maximum expansion of CFU-GM (colony-forming unit granulocytemacrophage) cells was observed on day 10 for all cultures. Perfusion cultures had a maximum CFU-GM expansion of 17- and 19-fold with and without a stromal layer, respectively. In contrast, static cultures had a maximum CFU-GM expansion of 18- and 13-fold with and without a stromal layer, respectively. Average long-term-culture initiating cell (LTC-IC) numbers on day 15 were 34{\%} and 64{\%} of input in stroma-containing and stromafree perfusion cultures and 12{\%} and 11{\%} of input in stroma-containing and stroma-free static cultures, respectively. Thus, perfusion enhanced CFU-GM expansion and LTC-IC maintenance more for the stroma-free cultures than for stroma-containing cultures. This was surprising because analysis of medium supernatants indicated that the stroma-containing cultures were metabolically more active than the stroma-free cultures. In view of their equivalent, if not superior, performance compared to stroma-containing cultures, stroma-free perfusion cultures may offer significant advantages for potential clinical applications.",
keywords = "Bone marrow stroma, Cell retention, Hematopoietic cultures, Mononuclear cell cultures, Perfusion chamber",
author = "Sandstrom, {Craig E.} and Bender, {James G.} and Miller, {William M} and Papoutsakis, {E. Terry}",
year = "1996",
month = "6",
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T1 - Development of novel perfusion chamber to retain nonadherent cells and its use for comparison of human "mobilized" peripheral blood mononuclear cell cultures with and without irradiated bone marrow stroma

AU - Sandstrom, Craig E.

AU - Bender, James G.

AU - Miller, William M

AU - Papoutsakis, E. Terry

PY - 1996/6/5

Y1 - 1996/6/5

N2 - Perfusion and static cultures of peripheral blood (PB) mononuclear cells (MNCs), obtained from patients following stem cell mobilization, were supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF) and compared with and without a preformed irradiated allogeneic bone marrow stromal layer. Perfusion cultures without a stromal layer effectively retained nonadherent cells through the use of a novel "grooved" perfusion chamber, which was designed with minimal mass transfer barriers in order to achieve a well-defined culture environment. The grooved chamber allowed easy and efficient culture inoculation and cell recovery. Average maximum expansion of CFU-GM (colony-forming unit granulocytemacrophage) cells was observed on day 10 for all cultures. Perfusion cultures had a maximum CFU-GM expansion of 17- and 19-fold with and without a stromal layer, respectively. In contrast, static cultures had a maximum CFU-GM expansion of 18- and 13-fold with and without a stromal layer, respectively. Average long-term-culture initiating cell (LTC-IC) numbers on day 15 were 34% and 64% of input in stroma-containing and stromafree perfusion cultures and 12% and 11% of input in stroma-containing and stroma-free static cultures, respectively. Thus, perfusion enhanced CFU-GM expansion and LTC-IC maintenance more for the stroma-free cultures than for stroma-containing cultures. This was surprising because analysis of medium supernatants indicated that the stroma-containing cultures were metabolically more active than the stroma-free cultures. In view of their equivalent, if not superior, performance compared to stroma-containing cultures, stroma-free perfusion cultures may offer significant advantages for potential clinical applications.

AB - Perfusion and static cultures of peripheral blood (PB) mononuclear cells (MNCs), obtained from patients following stem cell mobilization, were supplemented with interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF) and compared with and without a preformed irradiated allogeneic bone marrow stromal layer. Perfusion cultures without a stromal layer effectively retained nonadherent cells through the use of a novel "grooved" perfusion chamber, which was designed with minimal mass transfer barriers in order to achieve a well-defined culture environment. The grooved chamber allowed easy and efficient culture inoculation and cell recovery. Average maximum expansion of CFU-GM (colony-forming unit granulocytemacrophage) cells was observed on day 10 for all cultures. Perfusion cultures had a maximum CFU-GM expansion of 17- and 19-fold with and without a stromal layer, respectively. In contrast, static cultures had a maximum CFU-GM expansion of 18- and 13-fold with and without a stromal layer, respectively. Average long-term-culture initiating cell (LTC-IC) numbers on day 15 were 34% and 64% of input in stroma-containing and stromafree perfusion cultures and 12% and 11% of input in stroma-containing and stroma-free static cultures, respectively. Thus, perfusion enhanced CFU-GM expansion and LTC-IC maintenance more for the stroma-free cultures than for stroma-containing cultures. This was surprising because analysis of medium supernatants indicated that the stroma-containing cultures were metabolically more active than the stroma-free cultures. In view of their equivalent, if not superior, performance compared to stroma-containing cultures, stroma-free perfusion cultures may offer significant advantages for potential clinical applications.

KW - Bone marrow stroma

KW - Cell retention

KW - Hematopoietic cultures

KW - Mononuclear cell cultures

KW - Perfusion chamber

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M3 - Review article

VL - 50

SP - 493

EP - 504

JO - Biotechnology and Bioengineering

JF - Biotechnology and Bioengineering

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