FAQ
- 用途別細胞培養
- ヒト末梢血から分離したCD34陽性細胞を好中球に分化培養させる方法はありますか？
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CD34陽性細胞からの好中球分化に関しましては、STEMCELL Technologies社より下記の情報が得られています。造血幹細胞用無血清培地のStemSpan SFEMを利用した好中球分化の方法になります。
For inducing neutrophil differentiation from CD34+ cells one could recommend using StemSpan SFEM supplemented with StemSpan CC100 Cytokine Cocktail and adding G-CSF either immediately or upon replating after a few days. Cultures should be monitored closely for cell growth viability and phenotype as the outcome is dependent on many variables: cell purity, quality and density culture time refeeding schedules. The timing is important as it is easy to overgrow these cultures. Neutrophils are shortlived cells and cell yield or quality may suffer if culture conditions and timing are not right. Other cell types will also be produced specifically Macrophages and Erythroid cells (even in the absence of EPO). Some publications recommend a replating schedule with eventual switch to G-CSF only which is intended to promote selective outgrowth and maturation of Neutrophils only. It can take 14-16 days to achieve complete differentiation from CD34+ cells into Neutrophils.
JY, Kuan JY, Lonkar PS, Krause DS, Seidman MM, Peterson KR, Nielsen PE, Kole R, Glazer PM. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids. Proc Natl Acad Sci U S A. 105:13514-9, 2008.
MPB CD34+ cells differentiated into neutrophils. For their neutrophil differentiation protocol see Supporting Information (under Cell Transfection)
Cells were plated in StemSpan SFEM + CC100 cytokine cocktail (100 ng/mL Flt-3L 100 ng/mL SCF, 20 ng/mL IL-3, 20 ng/mL IL-6) immediately following neucleofection. 48 hours after neucleofection culture media was switched to SFEM with 100 ng/mL Flt-3L, 50 ng/mL SCF, 5 ng/mL IL-3, 5 ng/mL GM-CSF and 30 ng/mL G-CSF for days 2-5 following nucleofection. Media was again switched to SFEM with 5 ng/mL IL-3 and 30 ng/mL G-CSF for days 6-9 following nucleofection and thereafter 30 ng/mL G-CSF in SFEM for up to 14 days following nucleofection.
Hino M et al. Thrombopoietin stimulates ex vivo expansion of mature neutrophils in the early stages of differentiation. Ann Hematol. 2003 Nov;82(11):671-6. Epub 2003 Oct 3.
This group cultured CD34+ cells purified from PB in basal medium (IMDM) supplemented with FBS and SCF, IL-3, TPOand G-CSF for the first 7 days. For the later phase of culture (the following 9 days), cells were cultured in IMDM with FBS and G-CSF alone. The same cytokine combination and plating schedule could be attempted using StemSpan in place of IMDM + FBS.
Tatsumi N et al. Ex vivo expansion of mature human neutrophils with normal functions from purified peripheral blood CD34+ haematopoietic progenitor cells. Br J Haematol. 2000 May;109(2):314-21.
PB CD34+ cells were cultured in RPMI-1640 medium supplemented with 10% FCS, SCF, IL-3, GM-CSF and G-CSF for 7 d and were further cultivated for another 7 d with G-CSF alone to achieve cultures with 95% functional neutrophils. The same cytokine combination and plating schedule could be attempted using StemSpan in place of RPMI + FBS.
Flavia Marturana* Nicholas E. Timmins* & Lars K. Nielsen. Short-term exposure of umbilical cord blood CD34+ cells to granulocyte–macrophage colony-stimulating factor early in culture improves ex vivo expansion of neutrophils. Cytotherapy 2011: 366-377.
Adrian Schreiber et al. Membrane Proteinase 3 Expression in Patients with Wegener’s Granulomatosis and in Human Hematopoietic Stem Cell–Derived Neutrophils. JASN July 1 2005 vol. 16 no. 7 2216-2224.
利用している培地製品および成長因子は下記のものになります。
StemSpan SFEM (ST-09600,ST-09650)
StemSpan CC100 (ST-02690)
Human G-CSF (ST-02615)
