In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood

H. Siti Norhaiza, Rohaya Megat Abdul Wahab, Z. A Intan Zarina, Shahrul Hisham Zainal Ariffin

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin +) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin - to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin- stem cells for at least 18 days. The Lin - stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin- stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.

Original languageEnglish
Title of host publicationAIP Conference Proceedings
Pages238-245
Number of pages8
Volume1571
DOIs
Publication statusPublished - 2013
Event2013 UKM Faculty of Science and Technology Post-Graduate Colloquium - Selangor
Duration: 3 Jul 20134 Jul 2013

Other

Other2013 UKM Faculty of Science and Technology Post-Graduate Colloquium
CitySelangor
Period3/7/134/7/13

Fingerprint

stem cells
blood
expansion
cells
interleukins
blood cells
cell culturing
pens
calves
macrophages
exclusion
serums
markers
therapy
disorders
low frequencies
gradients

Keywords

  • Expansion
  • Lineage negative
  • Lineage positive
  • Mononuclear cells
  • Peripheral blood

ASJC Scopus subject areas

  • Physics and Astronomy(all)

Cite this

In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood. / Norhaiza, H. Siti; Megat Abdul Wahab, Rohaya; Zarina, Z. A Intan; Zainal Ariffin, Shahrul Hisham.

AIP Conference Proceedings. Vol. 1571 2013. p. 238-245.

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Norhaiza, HS, Megat Abdul Wahab, R, Zarina, ZAI & Zainal Ariffin, SH 2013, In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood. in AIP Conference Proceedings. vol. 1571, pp. 238-245, 2013 UKM Faculty of Science and Technology Post-Graduate Colloquium, Selangor, 3/7/13. https://doi.org/10.1063/1.4858661
Norhaiza, H. Siti ; Megat Abdul Wahab, Rohaya ; Zarina, Z. A Intan ; Zainal Ariffin, Shahrul Hisham. / In vitro expansion of Lin+ and Lin- mononuclear cells from human peripheral blood. AIP Conference Proceedings. Vol. 1571 2013. pp. 238-245
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abstract = "Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin +) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin - to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10{\%} (v/v) Newborn Calf Serum (NBCS)+ 2{\%} (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin- stem cells for at least 18 days. The Lin - stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin- stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.",
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N2 - Haematopoietic stem cells (HSCs) are used in the therapy of blood disorders due to the ability of these cells to reconstitute haematopoietic lineage cells when transplanted into myeloablative recipients. However, substantial number of cells is required in order for the reconstitution to take place. Since HSCs present in low frequency, larger number of donor is required to accommodate the demand of transplantable HSCs. Therefore, in vitro expansion of HSCs will have profound impact on clinical purposes. The aim of this study was to expand lineage negative (Lin-) stem cells from human peripheral blood. Total peripheral blood mononuclear cells (PBMNCs) were fractionated from human blood by density gradient centrifugation. Subsequently, PBMNCs were subjected to magnetic assisted cell sorter (MACS) which depletes lineage positive (Lin +) mononuclear cells expressing lineage positive markers such as CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, and CD235a to obtained Lin- cell population. The ability of Lin+ and Lin - to survive in vitro was explored by culturing both cell populations in complete medium consisting of Alpha-Minimal Essential Medium (AMEM) +10% (v/v) Newborn Calf Serum (NBCS)+ 2% (v/v) pen/strep. In another experiment, Lin+ and Lin- were cultured with complete medium supplemented with 10ng/mL of the following growth factors: stem cell factor (SCF), interleukin (IL)-3, granulocyte-macrophage colony stimulating factor (GM-CSF), 2IU/mL of Erythropoietin (Epo) and 20ng/mL of IL-6. Three samples were monitored in static culture for 22 days. The expansion potential was assessed by the number of total viable cells, counted by trypan blue exclusion assay. It was found that Lin+ mononuclear cells were not able to survive either in normal proliferation medium or proliferation medium supplemented with cytokines. Similarly, Lin- stem cells were not able to survive in proliferation medium however, addition of cytokines into the proliferation medium support Lin- stem cells for at least 18 days. The Lin - stem cells started to response to the cytokines added as early as Day 2 of culture. It is concluded that Lin- stem cells can be expanded in vitro by culturing in proliferation medium supplemented with cytokines.

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KW - Expansion

KW - Lineage negative

KW - Lineage positive

KW - Mononuclear cells

KW - Peripheral blood

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